Methods to determine immunogenicity of humanized anti-tag 72 cc49 antibodies

ABSTRACT

The present disclosure provides humanized CC49 monoclonal antibodies that bind TAG-72 with high binding affinity and that are minimally immunogenic. In one embodiment, a humanized CC49 antibody includes a non-conservative amino acid substitution in a light chain complementarity determining region 3 of the CC49 antibody. In a further embodiment, the humanized CC49 antibody includes a non-conservative substitution of a first residue in a light chain complementarity determining region 3 and a substitution of a second residue in a complementarity determining region of the humanized CC49 antibody. In several of the embodiments, methods are disclosed for the use of a humanized CC49 antibody.

PRIORITY CLAIM

This is a continuation of U.S. application Ser. No. 12/474,221, filedMay 28, 2009, which is a divisional of U.S. application Ser. No.10/519,580, filed Dec. 27, 2004, now U.S. Pat. No. 7,569,673, issuedAug. 4, 2009, which is the §371 U.S. National Stage of InternationalApplication No. PCT/US2003/020367, filed Jun. 26, 2003, which waspublished in English under PCT Article 21 (2), which in turn claims thebenefit of U.S. Provisional Application No. 60/393,077, filed Jun. 28,2002. The entire disclosure of each of these applications is herebyexpressly incorporated by reference.

FIELD

The present disclosure relates to humanized monoclonal antibodies thatbind a tumor antigen. More specifically, the present disclosure relatesto humanized monoclonal antibodies with non-conservative amino acidsubstitutions that have a high binding affinity for tumor-associatedglycoprotein (TAG)-72 and minimal immunogenicity.

BACKGROUND

The use of murine monoclonal antibodies in medicine has significantpotential especially in the diagnosis and treatment of various diseases,including cancer. The advantage of using monoclonal antibodies residesin their specificity for a single antigen. A monoclonal antibody raisedagainst a specific tumor cell surface antigen can be coupled totherapeutic agents, such as radioisotopes and chemotherapeutic drugs,and these immunoconjugates can be used clinically to specificallytarget, for example, a tumor cell of interest.

A major limitation in the clinical use of monoclonal antibodies is thedevelopment of a human anti-murine antibody (HAMA) response in thepatients receiving the treatments. The HAMA response can involveallergic reactions and an increased rate of clearance of theadministered antibody from the serum. Various types of modifiedmonoclonal antibodies have been developed to minimize the HAMA responsewhile trying to maintain the antigen binding affinity of the parentmonoclonal antibody. One type of modified monoclonal antibody is ahuman-mouse chimera in which a murine antigen-binding variable region iscoupled to a human constant domain (Morrison and Schlom, ImportantAdvances in Oncology, Rosenberg, S. A. (Ed.), 1989). A second type ofmodified monoclonal antibody is the complementarity determining region(CDR)-grafted, or humanized, monoclonal antibody (Winter and Harris,Immunol. Today 14:243-246, 1993).

The tumor-associated glycoprotein (TAG)-72, is expressed on the cells ofa majority of human carcinomas, including adenocarcinoma, colorectal,gastric, pancreatic, breast, lung and ovarian carcinomas. Murinemonoclonal antibodies have been disclosed that specifically bind TAG-72.One of these antibodies, CC49, has been shown to efficiently target andreduce the size of human colon carcinoma xenografts in nude mice, andhas been targeted to a variety of carcinomas in a number of clinicaltrials. Unfortunately, the clinical utility of the CC49 monoclonalantibody has been limited because of its murine origin. Thus, thereclearly exists a need to develop a humanized CC49 antibody with bothhigh antigen binding affinity and low immunogenicity for use in humansubjects.

SUMMARY

The present disclosure relates to humanized CC49 monoclonal antibodiesthat bind TAG-72 with high binding affinity and that are minimallyimmunogenic.

In one embodiment of the disclosure, a humanized CC49 antibody includesa non-conservative amino acid substitution in a light chaincomplementarity determining region 3 of the CC49 antibody, or functionalfragment thereof, and has a high binding affinity for TAG-72.

In another embodiment, a humanized CC49 antibody includes a nucleic acidsequence encoding the antibody that is deposited as ATCC Accessionnumber PTA-4182 or ATCC Accession number PTA-4183.

In one embodiment, a humanized CC49 antibody with high binding affinityfor TAG-72 and minimal immunogenicity includes a variable lightframework region and a variable heavy framework region of a humanantibody. The humanized CC49 antibody has at least one complementaritydetermining region from a human antibody and the remainingcomplementarity determining regions from a murine CC49 antibody. Thehumanized CC49 antibody also includes a non-conservative substitution ofa first residue in a light chain complementarity determining region 3and a substitution of a second residue in a complementarity determiningregion of the human CC49 antibody.

Methods are disclosed herein for the use of a humanized CC49 antibody inthe detection or treatment of a tumor in a subject. In one specificembodiment, a method is disclosed for detecting a tumor. The methodincludes contacting a sample obtained from the subject with a humanizedCC49 antibody for a sufficient amount of time to form an immune complex,and then detecting the presence of the immune complex. Another method isdisclosed for detecting a tumor in a subject that includes administeringa humanized CC49 antibody to the subject for a sufficient amount of timeto form an immune complex and then detecting the presence of the immunecomplex. In a further embodiment, a method is disclosed for treating asubject having a tumor that expresses TAG-72. The method includesadministering to the subject a therapeutically effective amount of ahumanized CC49 antibody, for example, such as an antibody conjugated toa drug or toxin.

A kit is disclosed herein that includes a container with the humanizedCC49 antibody described herein.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic drawing comparing amino acid substitutions inCC49, HuCC49V10, HuCC49V10-14 and HuCC49V10-15. Amino acid residuenumber is shown at the top of the figure. CDR region is indicated at thebottom of the figure.

FIG. 2 is a schematic representation of the phage display vector,PComb3H-SS. Only those restriction endonuclease sites are shown that arerelevant to cloning of the target genes or converting the expressionconstruct for soluble Fab expression. Gene III indicates a sequenceencoding the carboxyl-terminal domain of the gene III protein of phageM13. Lac Z p represents the lac Z promoter of E. coli. Omp A and pel Bare the prokaryotic leader sequences; RBS shows ribosomal binding sitefor protein translation. SS I and SS II are the stuffer sequences, andstop denotes the termination codon for protein synthesis.

FIG. 3 is a schematic representation of the vectors pIZ/V5-His (FIG. 3A)and ppIB/V5-His (FIG. 3B) for the expression of proteins in insectcells.

FIG. 4 is a set of digital images demonstrating SDS-PAGE analysis ofpurified HuCC49 and the variant antibodies derived from it undernon-reducing (FIG. 4A) and reducing (FIG. 4B) conditions. Lane 1,HuCC49; lane 2, HuCC49V10; lane 3, HuCC49V10-7; lane 4, HuCC49V10-12,lane 5, HuCC49V10-14, lane 6, HuCC49V10-15 (lane designations are thesame for FIGS. 4A and 4B).

FIG. 5 is a graph demonstrating the reactivity of CC49 antibodies(identified by their symbols in the inset) in a competition RIA.Increasing concentrations of different antibodies were used to competefor the binding of ¹²⁵I-labeled HuCC49 to the TAG-72 positive BSM.

FIG. 6 is a series of tables of flow cytometric analysis of the bindingof CC49 derived recombinant antibodies to Jurkat cells that expressTAG-72 antigen on their cell surface. The percent of gated cells fordifferent antibodies are tabulated.

FIG. 7 is a set of graphs demonstrating the reactivity of HuCC49 and itsvariants to sera from patient EA (FIG. 7A) and DS (FIG. 7B), measured bysurface plasma resonance (SPR). Increasing concentrations of theantibodies tested were used to compete with the sera anti-idiotypic(anti variable region) antibodies for binding to HuCC49 immobilized onsensor chip. Percent binding of the sera to HuCC49 was calculated fromthe sensogram and plotted as a function of the concentration of thecompetitor.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file4239_(—)66176_(—)07_Sequence_Listing, Feb. 28, 2011, 4.19 KB, which isincorporated by reference herein.

DETAILED DESCRIPTION I. Abbreviations

-   BSM bovine submaxillary mucin-   C constant-   CH constant heavy-   CL constant light-   CDR complementarity determining region-   Fab fragment antigen binding-   F(ab′)₂ Fab with additional amino acids, including cysteines    necessary for disulfide bonds-   FACS fluorescence activated cell sort-   FR framework region-   Fv fragment variable-   H heavy-   HAMA human antimurine antibody-   HuIgG human immunoglobulin G-   Ig immunoglobulin-   Ka relative affinity constant-   L light-   PCR polymerase chain reaction-   scFv single chain Fv-   SDR specificity determining residue-   SPR surface plasmon resonance-   TAG-72 tumor associated glycoprotein-72-   V variable-   VH variable heavy-   VL variable light

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: A Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: Immunoglobulin (Ig) molecules and immunologically activeportions of Ig molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen. Inone embodiment the antigen is tumor-associated glycoprotein (TAG-72).Monoclonal, and humanized immunoglobulins are encompassed by thedisclosure. In one embodiment, a murine monoclonal antibody thatrecognizes the TAG-72 antigen is CC49. In another embodiment, ahumanized CC49 antibody is HuCC49. In other embodiments, varianthumanized CC49 antibodies are HuCC49V10-14 or HuCC49V10-15. Thedisclosure also includes synthetic and genetically engineered variantsof these immunoglobulins.

A naturally occurring antibody (e.g., IgG) includes four polypeptidechains, two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. However, it has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a naturallyoccurring antibody. Thus, these antigen-binding fragments are alsointended to be designated by the term “antibody.” Examples of bindingfragments encompassed within the term antibody include (i) an Fabfragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fdfragment consisting of the VH and CH1 domains; (iii) an Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (iv)a dAb fragment (Ward et al., (1989) Nature 341:544-546) which consistsof a VH domain; and (v) an F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion. Furthermore, although the two domains of the Fv fragment arecoded for by separate genes, a synthetic linker can be made that enablesthem to be made as a single protein chain (known as single chain Fv(scFv); Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. 85:5879-5883) by recombinant methods. Such singlechain antibodies, as well as dsFv, a disulfide stabilized Fv (Bera etal. (1998) J. Mol. Biol. 281:475-483), and dimeric Fvs (diabodies), thatare generated by pairing different polypeptide chains (Holliger et al.(1993) Proc. Natl. Acad. Sci. 90:6444-6448), are also included.

In one embodiment, antibody fragments for use in this disclosure arethose which are capable of cross-linking their target antigen, e.g.,bivalent fragments such as F(ab′)₂ fragments. Alternatively, an antibodyfragment which does not itself cross-link its target antigen (e.g., aFab fragment) can be used in conjunction with a secondary antibody whichserves to cross-link the antibody fragment, thereby cross-linking thetarget antigen. Antibodies can be fragmented using conventionaltechniques and the fragments screened for utility in the same manner asdescribed for whole antibodies. An antibody is further intended toinclude humanized monoclonal molecules that specifically bind the targetantigen.

“Specifically binds” refers to the ability of individual antibodies tospecifically immunoreact with an antigen. This binding is a non-randombinding reaction between an antibody molecule and the antigen. In oneembodiment, the antigen is TAG-72. Binding specificity is typicallydetermined from the reference point of the ability of the antibody todifferentially bind the antigen of interest and an unrelated antigen,and therefore distinguish between two different antigens, particularlywhere the two antigens have unique epitopes. An antibody thatspecifically binds to a particular epitope is referred to as a “specificantibody.”

A variety of methods for linking effector molecules to antibodies arewell known in the art. Detectable labels useful for such purposes arealso well known in the art, and include radioactive isotopes such as³²P, fluorophores, chemiluminescent agents, and enzymes. Alsoencompassed in the disclosure are the chemical or biochemicalmodifications that incorporate toxins in the antibody. In oneembodiment, the toxin is chemically conjugated to the antibody. Inanother embodiment, a fusion protein is genetically engineered toinclude the antibody and the toxin. Specific, non-limiting examples oftoxins are radioactive isotopes, chemotherapeutic agents, bacterialtoxins, viral toxins, or venom proteins. The disclosure also includeschemical or genetically engineered modifications that link a cytokine toan antibody (such as by a covalent linkage). Specific, non-limitingexamples of cytokines are interleukin (IL)-2, IL-4, IL-10, tumornecrosis factor (TNF)-alpha and interferon (IFN)-gamma.

Antigen: Any molecule that can bind specifically with an antibody. Anantigen is also a substance that antagonizes or stimulates the immunesystem to produce antibodies. Antigens are often foreign substances suchas allergens, bacteria or viruses that invade the body.

CC49 monoclonal antibody: A murine monoclonal antibody of the IgG₁isotype that specifically binds TAG-72 (deposited as ATCC Accession No.HB 9459). This monoclonal antibody is a second generation monoclonalantibody prepared by immunizing mice with TAG-72 that was purified usingthe first generation antibody B72.3 (Colcher et al., Proc. Natl. Acad.Sci. USA 78:3199-3203, 1981). The CC49 monoclonal antibody efficientlytargets human colon carcinoma xenografts in athymic mice and reduces oreliminates their growth (Colcher et al., Cancer Res. 48:4597-4603,1988). Radiolabeled CC49 has been shown to successfully target a numberof human tumors including adenocarcinoma, colorectal, breast, prostateand ovarian (Liu et al., Cancer Biotherap Radiopharm. 12:79-87, 1997;Macey et al., Clin. Cancer Res. 3:1547-1555, 1997; Meredith et al. J.Nucl. Med., 37:1491-1496, 1996.)

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Chimeric antibody: An antibody which includes sequences derived from twodifferent antibodies, which typically are of different species. Mosttypically, chimeric antibodies include human and murine antibodydomains, generally human constant and murine variable regions.

Complementarity Determining Region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of an Igeach have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1,H-CDR2, H-CDR3, respectively. By definition, the CDRs of the light chainare bounded by the residues at positions 24 and 34 (L-CDR1), 50 and 56(L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain are bounded bythe residues at positions 31 and 35b (H-CDR1), 50 and 65 (H-CDR2), 95and 102 (H-CDR3), using the numbering convention delineated by Kabat etal., (1991) Sequences of Proteins of Immunological Interest, 5^(th)Edition, Department of Health and Human Services, Public Health Service,National Institutes of Health, Bethesda (NIH Publication No. 91-3242).

Constant Region: The portion of the antibody molecule which conferseffector functions. In the present disclosure, the variant antibodiesinclude constant regions derived from human immunoglobulins. The heavychain constant region can be selected from any of five isotypes: alpha,delta, epsilon, gamma or mu. Heavy chains of various subclasses (such asthe IgG subclass of heavy chains) are responsible for different effectorfunctions. Thus, by choosing the desired heavy chain constant region,humanized antibodies with the desired effector function can be produced.The light chain constant region can be of the kappa or lambda type.

Cytotoxin: An agent that is toxic for cells. Examples of cytotoxinsinclude radioactive isotopes, chemotherapeutic drugs, bacterial toxins,viral toxins, and proteins contained in venom (e.g. insect, reptile, oramphibian venom). A cytokine, such as interleukin-2 or interferon, canalso be a cytotoxin.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer whichconstitutes the genetic material of most living organisms (some viruseshave genes composed of ribonucleic acid (RNA)). The repeating units inDNA polymers are four different nucleotides, each of which contains oneof the four bases, adenine, guanine, cytosine and thymine bound to adeoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides (referred to as codons) code for each amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequence of three nucleotides in the mRNA that istranscribed from the DNA.

Effector Molecule: Therapeutic, diagnostic or detection moieties linkedto an antibody, using any number of means known to those of skill in theart. Both covalent and noncovalent linkage means may be used. Theprocedure for linking an effector molecule to an antibody variesaccording to the chemical structure of the effector. Polypeptidestypically contain a variety of functional groups; e.g., carboxylic acid(COOH), free amine (—NH₂) or sulfhydryl (—SH) groups, which areavailable for reaction with a suitable functional group on an antibodyto result in the linkage of the effector molecule. Alternatively, theantibody is derivatized to expose or link additional reactive functionalgroups. The derivatization may involve linkage of any of a number oflinker molecules such as those available from Pierce Chemical Company,Rockford Ill. The linker can be any molecule used to join the antibodyto the effector molecule. The linker is capable of forming covalentbonds to both the antibody and to the effector molecule. Suitablelinkers are well known to those of skill in the art and include, but arenot limited to, straight or branched-chain carbon linkers, heterocycliccarbon linkers, or peptide linkers. Where the antibody and the effectormolecule are polypeptides, the linkers may be joined to the constituentamino acids through their side groups (e.g., through a disulfide linkageto cysteine) or to the alpha carbon amino and carboxyl groups of theterminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used.

In view of the large number of methods that have been reported forlinking a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (e.g. enzymes or fluorescent molecules) drugs, toxins,and other agents to antibodies one skilled in the art will be able todetermine a suitable method for linking a given agent to an antibody.

Encode: A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Epitope: A site on an antigen recognized by an antibody, as determinedby the specificity of the antibody amino acid sequence. Epitopes arealso called antigenic determinants.

Framework Region: Amino acid sequences interposed between CDRs. Includesvariable light and variable heavy framework regions. The frameworkregions serve to hold the CDRs in an appropriate orientation for antigenbinding.

High binding affinity: Affinity of an antibody for an antigen where therelative affinity of the humanized CC49 antibody is significantlygreater than that of a parent CC49 antibody, for example HuCC49V10. Inone embodiment, affinity is calculated by a modification of theScatchard method described by Frankel et al. (1979) Mol. Immunol.,16:101-106. One of skill in the art can readily identify a statisticaltest that determines a statistically significant result for example, theStudent's t-test, the Wilcoxon two sample test, or the Median test. Inone embodiment, a high binding affinity is at least about 1.2×10⁻⁸ M. Inother embodiments, a high binding affinity is at least about 1.5×10⁻⁸,at least about 2.0×10⁻⁸, at least about 2.5×10⁻⁸, at least about3.0×10⁻⁸, at least about 3.5×10⁻⁸, at least about 4.0×10⁻⁸, at leastabout 4.5×10⁻⁸, or at least about 5.0×10⁻⁸ M.

In another embodiment, a high binding affinity is measured by anantigen/antibody dissociation rate of a humanized CC49 antibody that issignificantly lower than the parent CC49 antibody. In yet anotherembodiment, a high binding affinity is measured by a competitionradioimmunoassay, where the amount of antibody needed for 50% inhibitionof the binding of ¹²⁵I-labeled HuCC49 antibody to BSM is less than thatrequired by the parent CC49 antibody. In another embodiment, a highbinding affinity is measured by flow cytometry as an increased number ofgated cells labeled with humanized CC49 antibody compared to the numberof cells labeled by the parent CC49 antibody.

HAMA (Human anti-murine antibody) response: An immune response in ahuman subject to the variable and constant regions of a murine antibodythat has been administered to the patient. Repeated antibodyadministration may lead to an increased rate of clearance of theantibody from the patient's serum and may also elicit allergic reactionsin the patient.

Humanized antibody: A human antibody genetically engineered to includemouse hypervariable regions. In one embodiment, the DNA encodinghypervariable loops of mouse monoclonal antibodies or variable regionsselected in phage display libraries is inserted into the frameworkregions of human Ig genes. Antibodies can be “customized” to have adesired binding affinity or to be minimally immunogenic in the humanstreated with them.

Humanized CC49 antibodies: CC49 antibodies humanized by grafting CC49CDRs onto the frameworks of the relevant human antibodies (Kashmiri etal., Hybridoma, 14: 461-473, 1995). The murine CDRs in the resultanthumanized CC49 (HuCC49) could evoke an anti-idiotypic response whenadministered in human subjects. CC49 can be humanized by grafting onlyCC49 CDRs that are important for antigen binding onto the variable lightand variable heavy framework regions of, for example, LEN and 21/28′CLhuman antibodies (Tamura et al., J. Immunol. 164:1432-1441, 2000; WO00/26394). In addition, non-specificity determining residues (SDRs) inthe murine CDRs can be substituted with the corresponding residue in thehuman antibody. One specific, non-limiting example of a humanized CC49monoclonal antibody is HuCC49V10 (see published PCT patent applicationPCT/US99/25552, herein incorporated by reference). In one embodiment,HuCC49V10 has minimal immunogenicity (compared to the parental HuCC49antibody, at least 16-fold higher molar concentration of HuCC49V10 wasrequired to attain 25% inhibition of HuCC49 binding to patient serum)and a partial loss in antigen-binding affinity (1.15×10⁻⁸M) compared tothe parent HuCC49 antibody (3.20×10⁻⁸ M). In one embodiment, a humanizedCC49 antibody is HuCC49V10-14 (ATCC Accession Number PTA-4182, see FIG.1; also termed HuCC49V14 in the deposit). In another embodiment, ahumanized CC49 antibody is HuCC49V10-15 (ATCC Accession Number PTA-4183,see FIG. 1; also termed HuCC49V15 in the deposit).

Idiotype: the property of a group of antibodies or T cell receptorsdefined by their sharing a particular idiotope (an antigenic determinanton the variable region); i.e., antibodies that share a particularidiotope belong to the same idiotype. “Idiotype” may be used to describethe collection of idiotopes expressed by an Ig molecule. An“anti-idiotype” antibody may be prepared to a monoclonal antibody bymethods known to those of skill in the art and may be used to preparepharmaceutical compositions.

Immune cell: Any cell involved in a host defense mechanism. These caninclude, for example, T cells, B cells, natural killer cells,neutrophils, mast cells, macrophages, antigen-presenting cells,basophils, eosinophils, and neutrophils.

Immune response: A response of a cell of the immune system, such as aneutrophil, a B cell, or a T cell, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In another embodiment, the response is against an antibody,such as HAMA response, including an anti-variable region response.

Immunoconjugate: A covalent linkage of an effector molecule to anantibody. The effector molecule can be a toxin or a detectable label.Specific, non-limiting examples of toxins include, but are not limitedto, abrin, ricin, Pseudomonas exotoxin (such as PE35, PE37, PE38, andPE40), diphtheria toxin, anthrax toxin, botulinum toxin, or modifiedtoxins thereof. For example, Pseudomonas exotoxin and diphtheria toxinare highly toxic compounds that typically bring about death throughliver toxicity. Pseudomonas exotoxin and diphtheria toxin, however, canbe modified into a form for use as an immunotoxin by removing the nativetargeting component of the toxin (e.g., domain Ia of Pseudomonasexotoxin and the B chain of diphtheria toxin) and replacing it with adifferent targeting moiety, such as an antibody. Other toxic agents,that directly or indirectly inhibit cell growth or kill cells, includechemotherapeutic drugs, cytokines, for example interleukin-2 orinterferon, radioactive isotopes, viral toxins, or proteins containedwithin, for example, insect, reptile, or amphibian venom. Specific,non-limiting examples of detectable labels include, but are not limitedto, radioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent agents, fluorescent agents, haptens, or enzymes. A“chimeric molecule” is a targeting moiety, such as a ligand or anantibody, conjugated (attached or coupled) to an effector molecule. Theterm “conjugated” or “linked” refers to making two polypeptides into onecontiguous polypeptide molecule. In one embodiment, an antibody isjoined to an effector molecule. In another embodiment, an antibodyjoined to an effector molecule is further joined to a lipid or othermolecule to a protein or peptide to increase its half-life in theantibody. The linkage can be, for example, either by chemical orrecombinant means. In one embodiment, the linkage is chemical, wherein areaction between the antibody moiety and the effector molecule hasproduced a covalent bond formed between the two molecules to form onemolecule. A peptide linker (short peptide sequence) can optionally beincluded between the antibody and the effector molecule.

Immunogenicity: A measure of the ability of a targeting protein ortherapeutic moiety to elicit an immune response (humoral or cellular)when administered to a subject.

Immunoreactivity: A measure of the ability of an Ig to recognize andbind to a specific antigen.

Isolated: An biological component (such as a nucleic acid, peptide orprotein) that has been substantially separated, produced apart from, orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins that have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, chemiluminescent tags, haptens, enzymatic linkages, andradioactive isotopes.

Ligand contact residue: A residue within a CDR that is involved incontact with a ligand or antigen. A ligand contact residue is also knownas a specificity determining residue (SDR). A non-ligand contact residueis a residue in a CDR that does not contact a ligand. A non-ligandcontact residue can also be a framework residue.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B-cellsand T-cells.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Minimally immunogenic: An antibody that generates a reduced, for examplelow, immune response when administered to a subject, such as a humansubject. In one embodiment, immunogenicity is measured in a competitivebinding assay. In one specific, non-limiting example, immunogenicity isthe ability of a variant HuCC49 antibody to prevent a parental HuCC49antibody from binding to CC49 anti-idiotypic antibodies in a patient'sserum. If a variant HuCC49 antibody competes with an equal molar amountof the parental HuCC49 antibody (i.e. elicits greater than about 50%inhibition of parental HuCC49 binding to anti-idiotypic antibodies in apatient's serum) then the variant HuCC49 antibody is immunogenic. If avariant HuCC49 antibody competes poorly with an equal molar or lessamount of the parental HuCC49 antibody (i.e. elicits about 50% or lessinhibition of parental HuCC49 binding to anti-idiotypic antibodies in apatient's serum) then the variant HuCC49 antibody is minimallyimmunogenic. In another embodiment, if a five-fold or greater molarconcentration of a variant HuCC49 antibody is required to achieve about50% inhibition of binding of the parental antibody to its cognateanti-idiotypic antibodies present in a subject's sera, then the variantantibody is minimally immunogenic.

Monoclonal antibody: An antibody produced by a single clone ofB-lymphocytes. Monoclonal antibodies are produced by methods known tothose of skill in the art, for instance by making hybridantibody-forming cells from a fusion of myeloma cells with immune spleencells.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Oligonucleotide: A linear single-stranded polynucleotide sequence of upto about 200 nucleotide bases in length, for example a polymer ofdeoxyribonucleotides or ribonucleotides which is at least 6 nucleotides,for example at least 15, 50, 100 or even 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

Phage display: A technique wherein DNA sequences are amplified andcloned into phage vector to create a “phage library,” in which the phagepresent on their surface the proteins encoded by the DNA. In oneembodiment, a phage library is produced that expresses HuCC49V10 variantimmunoglobulins. From the rescued phages, the individual phage clonesare selected through interaction of the displayed protein with a ligand,and the specific phage is amplified by infection of bacteria. Antigenspecific immunoglobulins can then be expressed and characterized fortheir antigen binding and sera reactivity (potential immunogenicity).

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. “Incubating” includes a sufficientamount of time for a drug to interact with a cell. “Contacting” includesincubating a drug in solid or in liquid form with a cell.

A “therapeutically effective amount” is a quantity of a specificsubstance sufficient to achieve a desired effect in a subject beingtreated. For instance, this can be the amount necessary to inhibit orsuppress growth of a tumor or to decrease a sign or symptom of the tumorin the subject. In one embodiment, a therapeutically effective amount isthe amount necessary to eliminate a tumor. When administered to asubject, a dosage will generally be used that will achieve target tissueconcentrations (for example, in tumors) that has been shown to achieve adesired in vitro effect.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15^(th) Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of humanized CC49 monoclonalantibodies disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polynucleotide: A single-stranded linear nucleotide sequence, includingsequences of greater than 100 nucleotide bases in length.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred in nature. The termpolypeptide or protein as used herein encompasses any amino acidsequence and includes, but may not be limited to, modified sequencessuch as glycoproteins. The term polypeptide is specifically intended tocover naturally occurring proteins, as well as those that arerecombinantly or synthetically produced.

Substantially purified polypeptide as used herein refers to apolypeptide that is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

A non-conservative amino acid substitution can result from changes in:(a) the structure of the amino acid backbone in the area of thesubstitution; (b) the charge or hydrophobicity of the amino acid; or (c)the bulk of an amino acid side chain. Substitutions generally expectedto produce the greatest changes in protein properties are those inwhich: (a) a hydrophilic residue is substituted for (or by) ahydrophobic residue; (b) a proline is substituted for (or by) any otherresidue; (c) a residue having a bulky side chain, e.g., phenylalanine,is substituted for (or by) one not having a side chain, e.g., glycine;or (d) a residue having an electropositive side chain, e.g., lysyl,arginyl, or histadyl, is substituted for (or by) an electronegativeresidue, e.g., glutamyl or aspartyl.

Variant amino acid sequences may, for example, be 80, 90 or even 95 or98% identical to the native amino acid sequence. Programs and algorithmsfor determining percentage identity can be found at the NCBI website.

Preventing or treating a disease: Preventing a disease refers toinhibiting completely or in part the development or progression of adisease, for example in a person who is known to have a predispositionto a disease. An example of a person with a known predisposition issomeone with a history of cancer in the family, or who has been exposedto factors that predispose the subject to the development of a tumor.Treating a disease refers to a therapeutic intervention that inhibits,or suppressed the growth of a tumor, eliminates a tumor, ameliorates atleast one sign or symptom of a disease or pathological condition, orinterferes with a pathophysiological process, after the disease orpathological condition has begun to develop.

Protein: A biological molecule encoded by a gene and comprised of aminoacids.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or was made artificially. Artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques. Similarly, a recombinantprotein is one encoded by a recombinant nucleic acid molecule.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals.

TAG (Tumor-Associated Glycoprotein)-72: A cell-surface glycoprotein thatis expressed on human carcinomas, including adenocarcinoma, colorectal,gastric, pancreatic, breast, lung and ovarian carcinomas. TAG-72 has ahigh molecular weight (greater than 1×10⁶) as measured by size-exclusionchromatography, a density of 1.45 g/ml, is resistant to Chondroitinasedigestion, expresses blood group-related oligosaccharides, and isheavily sialylated with O-glycosidically linked oligosaccharidescharacteristic of mucins. These characteristics suggest that TAG-72 is amucin-like molecule (Johnson et al., Cancer Res. 46:850-857, 1986,incorporated herein by reference).

Therapeutically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit or suppress growthof a tumor. In one embodiment, a therapeutically effective amount is theamount necessary to eliminate a tumor. When administered to a subject, adosage will generally be used that will achieve target tissueconcentrations (for example, in tumors) that has been shown to achieve adesired in vitro effect.

Treatment: Refers to both prophylactic inhibition of initial infectionor disease, and therapeutic interventions to alter the natural course ofan untreated infection or disease process, such as a tumor growth or aninfection with a bacteria.

Tumor: A neoplasm that may be either malignant or non-malignant. Tumorsof the same tissue type are primary tumors originating in a particularorgan (such as breast, prostate, bladder or lung). Tumors of the sametissue type may be divided into tumor of different sub-types (a classicexample being bronchogenic carcinomas (lung tumors) which can be anadenocarcinoma, small cell, squamous cell, or large cell tumor). Breastcancers can be divided histologically into scirrhous, infiltrative,papillary, ductal, medullary and lobular. In one embodiment, cells in atumor express TAG-72.

Variable region (also variable domain or V domain): The regions of boththe light-chain and the heavy-chain on an Ig that containantigen-binding sites. The regions are composed of polypeptide chainscontaining four relatively invariant “framework regions” (FRs) and threehighly variant “hypervariable regions” (HVs). Because the HVs constitutethe binding site for antigen(s) and determine specificity by forming asurface complementarity to the antigen, they are more commonly termedthe “complementarity-determining regions,” or CDRs, and are denotedCDR1, CDR2, and CDR3. Because both of the CDRs from the heavy- andlight-chain domains contribute to the antigen-binding site, it is thethree-dimensional combination of the heavy and the light chain thatdetermines the final antigen specificity.

Within the heavy- and light-chain, the framework regions surround theCDRs. Proceeding from the N-terminus of a heavy or light chain, theorder of regions is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As used herein, theterm “variable region” is intended to encompass a complete set of fourframework regions and three complementarity-determining regions. Thus, asequence encoding a “variable region” would provide the sequence of acomplete set of four framework regions and threecomplementarity-determining regions.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means “including A or B, or A and B.” It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Humanized CC49 Antibodies

Disclosed herein are humanized monoclonal CC49 antibodies that have anon-conservative amino acid substitution in the light chaincomplementarity determining region (LCDR) 3 of the CC49 antibody graftedonto a human antibody framework. In one embodiment, the humanized CC49antibody has a non-conservative amino acid substitution of a ligandcontact residue in LCDR3. In several examples, the CC49 antibody has anon-conservative substitution of a ligand contact residue at position89, 90, 91, 92, 93, 94, 95 or 96 of LCDR3 (Table 1).

TABLE 1 HuCC49V10 CDR sequences LCDR3 89  90  91  92  93  94  95  96  97Gln Gln Tyr Tyr Ser Tyr Pro Leu Ser HCDR250  51  52   a  53  54  55  56  57 Tyr Phe Ser Pro Gly Asn Asp Asp PheHCDR2 58  59  60  61  62  63  64  65 Lys Tyr Ser Gln Lys Phe Gln Gly Inbold: The SDRs that are targeted for mutation. In italic: The residuesof HuCC49 already modified to generate HuCC49V10.

In one embodiment, the humanized CC49 antibody has a non-conservativeamino acid substitution at position 91 of LCDR3. In one specific,non-limiting example, the humanized CC49 antibody has a tyrosine toproline substitution at position 91 of LCDR3 (see HuCC49V10-14 in FIG. 1and in Table 2).

TABLE 2 Mutations in variants* isolated by phage display LCDR1 LCDR3 27b89 90 91 92 93 94 95 96 97 HuCC49V10 Val Gln Gln Tyr Tyr Ser Tyr Pro LeuSer HuCC49V10-7 Leu — — — — Leu — — — — HuCC49V10-10 — — — Ser — — — — —— HuCC49V10-12 — — — — — Leu — — — — HuCC49V10-13 — — — — — — Thr — — —HuCC49V10-14 — — — Pro — — — — — — HuCC49V10-15 Leu — — Pro — — — — — —HCDR2 50 51 52 a 53 54 55 56 57 HuCC49V10 Tyr Phe Ser Pro Gly Asn AspAsp Phe HuCC49V10-13 — — Asn — — — — — — HuCC49V10-7,   — — — — — — — —— -10, -12, -14, -15 HCDR2 (continued) 58 59 60 61 62 63 64 65 HuCC49V10Lys Tyr Ser Gln Lys Phe Gln Gly HuCC49V10-13 Gln — — — — — — —HuCC49V10-7,  — — — — — — — — -10, -12, -14, -15 Bold: The SDRs targetedfor mutation to generate a phage display library. Italic: The residuesof HuCC49 already modified to generate HuCC49V10. *Variants HuCC49V10-7,-10, -12,-13, -14, and -15 can be derived from HuCC49V10 as described inExamples 1-4,

In one embodiment, the humanized CC49 antibody has no more than onenon-conservative amino acid substitution in LCDR3. However, thehumanized CC49 antibody can include no more than two, no more thanthree, no more than four, no more than five, or no more than ninenon-conservative amino acid substitutions in LCDR3. In some embodiments,the CC49 antibody has a non-conservative amino acid substitution atposition 91, an additional non-conservative substitution of a ligandcontact residue at position 89, 90, 91, 92, 93, 94, 95 or 96 of LCDR3(Table 1), and has a high binding affinity for TAG-72. In anotherembodiment, the humanized CC49 antibody has a non-conservative aminoacid substitution at position 91, and has an additional non-conservativeamino acid substitution of a non-ligand contact residue of LCDR3.

A humanized CC49 monoclonal antibody including a non-conservative aminoacid substitution in LCDR3 can also include an additionalnon-conservative amino acid substitution in a region other than LCDR3.For example, an additional non-conservative amino acid substitution canbe a non-conservative substitution in another LCDR or in an HCDR.Specific, non-limiting examples of a humanized CC49 monoclonal antibodythat includes more than one non-conservative substitution are ahumanized CC49 monoclonal antibody with a non-conservative substitutionin an LCDR3 ligand contact residue and a non-conservative substitutionin an LCDR non-ligand contact residue, or a non-conservativesubstitution in LCDR3 and a non-conservative substitution in HCDR2. Inseveral embodiments, the CC49 antibody has a non-conservativesubstitution of a ligand contact residue at position 50, 51, 52, 52a,53, 54, 56, 57, or 58 of HCDR2 (Table 1) in addition to an LCDR3non-conservative substitution. In another embodiment, the humanized CC49antibody has an additional non-conservative amino acid substitution in aframework residue.

In one embodiment, the humanized CC49 antibody has a conservative aminoacid substitution in addition to an LCDR3 non-conservative substitution,such as, but not limited to, a conservative substitution in a CDR inaddition to an LCDR3 non-conservative substitution. In other specificnon-limiting examples, the humanized CC49 antibody has a conservativesubstitution in an LCDR or in an HCDR in addition to an LCDR3non-conservative substitution. In other specific non-limiting examples,the humanized CC49 antibody has a conservative substitution in LCDR1,LCDR2, LCDR3, HCDR1, HCDR2, or HCDR3 in addition to an LCDR3non-conservative substitution. Thus in another specific, non-limitingexample, the humanized CC49 antibody has a conservative amino acidsubstitution at position 27b of LCDR1 and a non-conservative amino acidsubstitution at position 91 of LCDR3. A specific, non-limiting exampleof a conservative amino acid substitution is a valine to leucinesubstitution at position 27b of LCDR1 and specific, non-limiting exampleof a non-conservative amino acid substitution is a tyrosine to prolinesubstitution at position 91 of LCDR3 (see HuCC49V10-15 in FIG. 1 and inTable 2).

In one embodiment, the humanized CC49 antibody has a CH2 domaindeletion. In one specific embodiment, a humanized CC49 antibody with aCH2 domain deletion is cleared more quickly from the plasma compared tothe parent CC49 antibody. In another specific embodiment, a humanizedCC49 antibody with a CH2 domain deletion has reduced immunogenicitycompared to the parent CC49 antibody.

The humanized monoclonal antibodies disclosed herein bind TAG-72 withhigh binding affinity. In one embodiment, the humanized CC49 antibodyhas a high binding affinity for TAG-72 that is at least about 1.2×10⁻⁸M. In other embodiments, the humanized CC49 antibody has a high bindingaffinity for TAG-72 that is at least about 1.2, ×10⁻⁸, about 1.5×10⁻⁸,about 2.0×10⁻⁸, about 2.5×10⁻⁸, about 3.0×10⁻⁸, about 3.5×10⁻⁸, about4.0×10⁻⁸, about 4.5×10⁻⁸, or about 5.0×10⁻⁸ M. In one embodiment, thehumanized CC49 antibody has a high binding affinity if it has asignificantly lower antigen/antibody dissociation rate compared to thatof the parent CC49 antibody. In another embodiment, the humanized CC49antibody has a high binding affinity if less antibody is required for a50% inhibition of the binding of ¹²⁵I-labeled HuCC49 to BSM compared tothe parent CC49 antibody. In yet another embodiment, the humanized CC49antibody has a high binding affinity when the number of cells labeledwith humanized CC49 antibody is significantly greater than the number ofcells labeled by the parent CC49 antibody, as measured by flowcytometry.

Immunogenicity of variant HuCC49 antibodies can be measured in acompetitive binding assay as the ability of a variant HuCC49 antibody toprevent a parent CC49 or HuCC49 antibody from binding to anti-idiotypicantibodies in a human subject's serum. In one embodiment, a varianthumanized CC49 antibody with a non-conservative amino acid substitutionin LCDR3 is minimally immunogenic in a subject. In other embodiments,the variant humanized CC49 antibody with an additional amino acidsubstitution is minimally immunogenic. In one embodiment, at least aboutfive-fold higher molar concentration of the variant humanized CC49antibody, than that of the parental HuCC49 antibody, is required toelicit 50% inhibition of the parental HuCC49 binding to its cognateanti-idiotypic antibodies in a subject's sera. In other embodiments, atleast about ten-fold, at least about twenty five-fold, at least aboutfifty-fold, at least about seventy-fold, or at least about onehundred-fold higher molar concentration of the variant humanized CC49antibody, than that of the parental antibody, is required to elicit 50%inhibition of the parental HuCC49 binding to its cognate anti-idiotypicantibodies in a subject's sera.

Effector molecules, e.g., therapeutic, diagnostic, or detectionmoieties, can be linked to a humanized CC49 antibody that specificallybinds TAG-72, using any number of means known to those of skill in theart. Thus, a humanized CC49 antibody with a non-conservative amino acidsubstitution can have any one of a number of different types of effectormolecules linked to it. In one embodiment, the humanized CC49 antibodyis linked to a detectable label. In some embodiments, the humanized CC49antibody is linked to a radioactive isotope, an enzyme substrate, aco-factor, a ligand, a chemiluminescent agent, a fluorescent agent, ahapten, or an enzyme. In another embodiment, the humanized CC49 antibodyis linked to a cytotoxin. In other embodiments, the humanized CC49antibody is linked to a chemotherapeutic drug, a radioactive isotope, abacterially-expressed toxin, a virally-expressed toxin, or a venomprotein. In yet other embodiments, the humanized CC49 antibody is linkedto a cytokine. Specific, non-limiting examples of cytokines are IL-2,IL-4, IL-10, TNF-alpha and IFN-gamma. In some embodiments, the humanizedCC49 antibody is linked to an effector molecule by a covalent ornon-covalent means.

Pharmaceutical Compositions and Therapeutic Methods

Pharmaceutical compositions are disclosed herein that include ahumanized CC49 monoclonal antibody, such as HuCC49V10-14 orHuCC49V10-15, and can be formulated with an appropriate solid or liquidcarrier, depending upon the particular mode of administration chosen. Inaddition, a humanized CC49 monoclonal antibody linked to an effectormolecule (i.e., toxin, chemotherapeutic drug, or detectable label) canbe prepared in pharmaceutical compositions.

The pharmaceutically acceptable carriers and excipients useful in thisdisclosure are conventional. For instance, parenteral formulationsusually comprise injectable fluids that are pharmaceutically andphysiologically acceptable fluid vehicles such as water, physiologicalsaline, other balanced salt solutions, aqueous dextrose, glycerol or thelike. Excipients that can be included are, for instance, other proteins,such as human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered can also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical, inhalation, oral and suppositoryformulations can be employed. Topical preparations can include eyedrops, ointments, sprays and the like. Inhalation preparations can beliquid (e.g., solutions or suspensions) and include mists, sprays andthe like. Oral formulations can be liquid (e.g., syrups, solutions orsuspensions), or solid (e.g., powders, pills, tablets, or capsules).Suppository preparations can also be solid, gel, or in a suspensionform. For solid compositions, conventional non-toxic solid carriers caninclude pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in the art.

The pharmaceutical compositions that include a humanized CC49 monoclonalantibody can be formulated in unit dosage form suitable for individualadministration of precise dosages. In addition, the pharmaceuticalcompositions may be administered as an immunoprophylactic in a singledose schedule or as an immunotherapy in a multiple dose schedule. Amultiple dose schedule is one in which a primary course of treatment maybe with more than one separate dose, for instance 1-10 doses, followedby other doses given at subsequent time intervals as needed to maintainor reinforce the action of the compositions. Treatment can involve dailyor multi-daily doses of compound(s) over a period of a few days tomonths, or even years. Thus, the dosage regime will also, at least inpart, be determined based on the particular needs of the subject to betreated and will be dependent upon the judgement of the administeringpractitioner. In one specific, non-limiting example, a unit dosage canbe about 0.1 to about 10 mg per patient per day. Dosages from about 0.1up to about 100 mg per patient per day may be used, particularly if theagent is administered to a secluded site and not into the circulatory orlymph system, such as into a body cavity, into a lumen of an organ, ordirectly into a tumor. In one embodiment, about 10 mCi of a radiolabeledhumanized CC49 monoclonal antibody is administered to a subject. Inother embodiments, about 15 mCi, about 20 mCi, about 50 mCi, about 75mCi or about 100 mCi of a radiolabeled humanized CC49 monoclonalantibody is administered to a subject. The amount of active compound(s)administered will be dependent on the subject being treated, theseverity of the affliction, and the manner of administration, and isbest left to the judgment of the prescribing clinician. Within thesebounds, the formulation to be administered will contain a quantity ofthe active component(s) in amounts effective to achieve the desiredeffect in the subject being treated.

The compounds of this disclosure can be administered to humans on whosetissues they are effective in various manners such as topically, orally,intravenously, intramuscularly, intraperitoneally, intranasally,intradermally, intrathecally, subcutaneously, via inhalation or viasuppository. The particular mode of administration and the dosageregimen will be selected by the attending clinician, taking into accountthe particulars of the case (e.g. the subject, the disease, the diseasestate involved, and whether the treatment is prophylactic).

In one embodiment, a therapeutically effective amount of a humanizedCC49 antibody, such as HuCC49V10-14 or HuCC49V10-15, is the amount ofhumanized CC49 antibody necessary to inhibit further growth of aTAG-72-expressing tumor or suppress the growth of a TAG-72-expressingtumor, without eliciting a HAMA response in the patient receiving thetreatment. In other embodiments, a therapeutically effective amount ofhumanized CC49 antibody is the amount of humanized CC49 antibodynecessary to eliminate or reduce the size of a TAG-72-expressing tumor,without eliciting a HAMA response. Specific, non-limiting examples ofTAG-72-expressing tumors are adenocarcinoma, colorectal, gastric,pancreatic, breast, lung, and ovarian tumors. In yet another embodiment,a therapeutically effective amount of humanized CC49 antibody is anamount of humanized CC49 antibody that is effective at reducing a signor a symptom of the tumor and induces a minimal immune response.

A therapeutically effective amount of a humanized CC49 monoclonalantibody, such as HuCC49V10-14 or HuCC49V10-15, can be administered in asingle dose, or in several doses, for example daily, during a course oftreatment. In one embodiment, treatment continues until a therapeuticresult is achieved. However, the effective amount of humanized CC49antibody will be dependent on the subject being treated, the severityand type of the affliction, and the manner of administration of thetherapeutic(s).

Controlled release parenteral formulations of a humanized CC49monoclonal antibody can be made as implants, oily injections, or asparticulate systems. For a broad overview of protein delivery systems(see Banga, A. J., Therapeutic Peptides and Proteins: Formulation,Processing, and Delivery Systems, Technomic Publishing Company, Inc.,Lancaster, Pa., 1995). Particulate systems include microspheres,microparticles, microcapsules, nanocapsules, nanospheres, andnanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres the therapeutic is dispersed throughoutthe particle. Particles, microspheres, and microcapsules smaller thanabout 1 μm are generally referred to as nanoparticles, nanospheres, andnanocapsules, respectively. Capillaries have a diameter of approximately5 μm so that only nanoparticles are administered intravenously.Microparticles are typically around 100 μm in diameter and areadministered subcutaneously or intramuscularly (see Kreuter, J.,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on ControlledDrug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,pp. 315-339, 1992).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, R., Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec et al., J. Parent. Sci. Tech.44:58, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri, et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa., 1993). Numerous additional systemsfor controlled delivery of therapeutic proteins are known (e.g., U.S.Pat. Nos. 5,055,303, 5,188,837, 4,235,871, 4,501,728, 4,837,0284,957,735 and 5,019,369, 5,055,303; 5,514,670; 5,413,797; 5,268,164;5,004,697; 4,902,505; 5,506,206, 5,271,961; 5,254,342 and 5,534,496).

Site-specific administration of the disclosed compounds can be used, forinstance by applying the humanized CC49 antibody to a pre-cancerousregion, a region of tissue from which a tumor has been removed, or aregion suspected of being prone to tumor development. In someembodiments, sustained intra-tumoral (or near-tumoral) release of thepharmaceutical preparation that includes a therapeutically effectiveamount of the humanized CC49 antibody may be beneficial.

The present disclosure also includes therapeutic uses of humanized CC49monoclonal antibodies that are non-covalently or covalently linked toeffector molecules. In one specific embodiment, the humanized CC49monoclonal antibody is covalently linked to an effector molecule that istoxic to a tumor or cell expressing TAG-72. In one specific,non-limiting example, the effector molecule is a cytotoxin. In otherspecific, non-limiting examples the effector molecule is a radioactiveisotope, a chemotherapeutic drug, a bacterially-expressed toxin, avirally-expressed toxin, a venom protein, or a cytokine. Humanized CC49monoclonal antibodies covalently linked to an effector molecule have avariety of uses. For example, a humanized CC49 antibody linked to aradioactive isotope us if use in immunotherapy. A humanized CC49antibody covalently linked to a radioactive isotope is of use tolocalize a tumor in radioimmunoguided surgery, such that the tumor canbe removed.

The present disclosure also includes combinations of a humanized CC49monoclonal antibody, such as HuCC49V10-14 or HuCC49V10-15, with one ormore other agents useful in the treatment of tumors. For example, thecompounds of this disclosure can be administered in combination witheffective doses of immunostimulants, anti-cancer agents,anti-inflammatory agents, anti-infectives, and/or vaccines. The term“administration in combination” or “co-administration” refers to bothconcurrent and sequential administration of the active agents. A subjectthat is suffering from a tumor, or is predisposed to the development ofa tumor, will be a candidate for treatment using the therapeutic methodsdisclosed herein.

Diagnostic Methods and Kits

A method is provided herein for the in vivo or in vitro detection ofTAG-72-expressing tumors or cells. An in vivo detection method canlocalize any tumor or cell that expresses TAG-72 in a subject. In oneembodiment, a humanized CC49 antibody is administered to the subject fora sufficient amount of time for the antibody to localize to the tumor orcell in the subject and to form an immune complex with TAG-72. Theimmune complex can then be detected. In one specific, non-limitingexample detection of an immune complex is performed byimmunoscintography. Other specific, non-limiting examples of immunecomplex detection include radiolocalization, radioimaging, orfluorescence imaging. In another embodiment, the antibody is linked toan effector molecule. In one specific, non-limiting embodiment, theeffector molecule is a detectable label. Specific, non-limiting examplesof detectable labels include a radioactive isotope, an enzyme substrate,a co-factor, a ligand, a chemiluminescent agent, a fluorescent agent, ahapten, or an enzyme.

A method of detecting tumors in a subject includes the administration ofa humanized CC49 antibody complexed to an effector molecule, such as aradioactive isotope. In one embodiment, a humanized CC49 antibodycomplexed to an effector molecule, such as a radioactive isotope, isadministered to a subject prior to surgery or treatment. In anotherembodiment, a humanized CC49 antibody complexed to an effector molecule,such as a radioactive isotope, is administered to a subject followingsurgery or treatment. After a sufficient amount of time has elapsed toallow for the administered radiolabeled antibody to localize to thetumor, the tumor is detected. In one specific embodiment, the detectionstep is performed prior to surgery. In another embodiment, the detectionstep is performed during surgery, for example to detect the location ofthe tumor prior to removing it, as in radioimmunoguided surgery. In yetanother embodiment, the detection step is performed after surgery toensure the complete removal of the tumor, or to detect a recurrence ofthe tumor. In one specific, non-limiting example, a radiolabeled immunecomplex is detected using a hand held gamma detection probe. Primarytumors, metastasized tumors or cells expressing TAG-72 can be detected.

In another embodiment, a humanized CC49 antibody and a secondaryantibody are administered to the subject for a sufficient amount of timefor the humanized CC49 antibody to form an immune complex with TAG-72 ona tumor or cell, and for the secondary antibody to form an immunecomplex with the humanized CC49 antibody. In one embodiment, thehumanized CC49 antibody is complexed with the secondary antibody priorto their administration to the subject. In one specific, non-limitingembodiment, the secondary antibody is linked to a detectable label. Inone embodiment, the immune complex, which includes TAG-72, the humanizedCC49 antibody, and the secondary antibody linked to a detectable label,is detected as described above.

An in vitro detection method can screen any biological sample containingany tumor or cell that expresses TAG-72. Such samples include, but arenot limited to, tissue from biopsies, autopsies, and pathologyspecimens. Biological samples also include sections of tissues, such asfrozen sections taken for histological purposes. Biological samplesfurther include body fluids, such as blood, serum, saliva, or urine. Abiological sample is typically obtained from a mammal, such as a human.In one embodiment the subject has a colorectal tumor. In otherembodiments, the subject has a gastric tumor, a pancreatic tumor, abreast tumor, a lung tumor, an adenocarcinoma, or an ovarian tumor.Other biological samples that can be detected by the in vitro detectionmethod include samples of cultured cells that express TAG-72.

In one embodiment, a method is provided for detecting aTAG-72-expressing tumor or cell. Kits for detecting a TAG-72-expressingtumor or cell will typically comprise a humanized CC49 antibody thatspecifically binds TAG-72. In some embodiments, an antibody fragment,such as an Fv fragment is included in the kit. In a further embodimentthe antibody is an immunoconjugate. In some embodiments, the antibody isconjugated to a detectable label (e.g. radioactive isotope, enzymesubstrate, co-factor, ligand, fluorescent agent, hapten, enzyme, orchemiluminescent agent).

The kit can include instructional materials disclosing means of use ofan antibody that specifically binds TAG-72 or fragment thereof (e.g. fordetection of TAG-72-expressing cells in a sample). The instructionalmaterials may be written, in an electronic form (e.g. computer disketteor compact disk) or may be visual (e.g. video files). The kits may alsoinclude additional components to facilitate the particular applicationfor which the kit is designed. Thus, for example, the kit mayadditionally contain means of detecting a label (e.g. enzyme substratesfor enzymatic labels, filter sets to detect fluorescent labels,appropriate secondary labels such as a secondary antibody, or the like).In one embodiment, the kit contains a secondary antibody that isconjugated to a detectable label. The kits may additionally includebuffers and other reagents, such as an antigen (e.g. purified TAG-72)routinely used for the practice of a particular method. Such kits andappropriate contents are well known to those of skill in the art.

In one embodiment of the present invention, the diagnostic kit comprisesan immunoassay. Although the details of the immunoassays may vary withthe particular format employed, the method of detecting TAG-72 orfragment thereof in a biological sample generally includes the steps ofcontacting the biological sample with an antibody which specificallyreacts, under immunologically reactive conditions, to TAG-72. Theantibody is allowed to specifically bind under immunologically reactiveconditions to form an immune complex, and the presence of the immunecomplex (bound antibody) is detected directly or indirectly.

The invention is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Construction of the Variant HuCC49V10 Antibody PhageLibrary

A phage display library of isolates was derived from the humanized CC49variant HuCC49V10 (Tamura et al., J. Immunol., 164: 1432, 2000) by themutagenesis of the LCDR3 and the HCDR2, the two CDRs that were earliershown to be the targets of the patient's anti-variable region response.Primer-induced mutagenesis was used to replace the targeted SDRs of thetwo CDRs (Table 1) with all possible residues located at thecorresponding positions in human antibodies (Table 3). A dual step PCR(Landt et al., Gene 96: 125, 1990) was used for DNA amplification.

TABLE 3 Residue substitutions in the library Encoded aa L-CDR3 Position89: R, L, N, K. M, H, Q, I, S Position 91: S, Y, R, A, G, H, T, C, P, D,N Positions 92, 93, 94, 96: S, R, L, V, A, G, T, P, Y, F, D, N, I, W, H,E, L, Q, M, C H-CDR2 Positions 50, 52, 53, 58: S, R, L, V, A, G, T, P,Y, F, D, N, I, W, H, E, L, Q, M, C Position 54: S, T, D, G, F, N, R, L,I, A, V, Y, C, H, P Position 56: S, T, A, G, V, L, R, I, D, E, Y, C, W,F, L, N, M,

DNA Amplification

For the first step of DNA amplification, a primer derived from theleader sequence of the light (SEQ ID NO: 1) or heavy (SEQ ID NO: 9)chain was used as the 5′ primer, while the degenerate mutagenic primersfor each of the light (SEQ ID NO: 2-7) or heavy (SEQ ID NO: 10-15) chainwere mixed together and used as 3′ primers (see Table 4 for primersequences of SEQ ID NO: 1-12). The degenerate primers were mixed in aratio that made all the 3′ primers equimolar in concentration. For thesecond step of the amplification, the gel purified product of the firstround of PCR served as the 5′ primer, while the 3′ primer was derivedfrom the 3′-end of the light chain (SEQ ID NO: 8) or the 3′-end of theCH1 region of the heavy chain (SEQ ID NO: 16).

TABLE 4Oligonucleotide primers used to generate the library of genes encoding light chainsand Fd regions of the variants of HuCC49V10 Name Sequence SEQ ID NO. 5′V_(L) 5′-TGAGCGGCACAGAGCTCGACATCGTGATGAG-3′ 1 3′ 89 V_(L)5′-AGCTATAATACTGSHKACAATAATAG-3′ 2 3′ 91 V_(L)5′-GGGGATAGCTATAGBNCTGCTGACAA-3′ 3 3′ 92 V_(L)5′-TGAGGGGATAGCTSNNATACTGCTGA-3′ 4 3′ 93 V_(L)5′-AGCTGAGGGGATASNNATAATACTGC-3′ 5 3′ 94 V_(L)5′-CGAAGCTGAGGGGSNNGCTATAATAC-3′ 6 3′ 96 V_(L)5′-CAGCGCCGAAGCTSNNGGGATAGCTA-3′ 7 3′ V_(L)5′-GCGCCGTCTAGAATTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCGAACTCAG-3′ 8 5′V_(H) 5′-GCCCGTACCATGGCCCAGGTCCAGCTGGTGCA-3′ 9 3′ 50 V_(H)5′-CGGGGAGAGAASNNTCCAATCCACT-3′ 10 3′ 52 V_(H)5′-CGTTTCCGGGSNNGAAATATCCAA-3′ 11 3′ 53 V_(H)5′-AATCATCGTTSNNGGGAGAGAAAT-3′ 12 3′ 54 V_(H)5′-AAAAATCATCGNNTCCGGGAGAGA-3′ 13 3′ 56 V_(H)5′-AGTACTTAAASNHATCGTTTCCGG-3′ 14 3′ 58 V_(H)5′-TCTGTGAGTASNNAAAATCATCGT-3′ 15 3′ V_(H)5′-GCATGTACTAGTTTTGCACAAGATTTGG-3′ 16 S = G/C H = A/C/T K = G/T B =G/T/C N = A/G/C/T

The oligonucleotide primers used for DNA amplification, listed in Table4, were supplied by Biosynthesis Inc. (Lewisville, Tex.). They werepurified by polyacrylamide gel electrophoresis. Each of the 5′ primersused for the first step PCR and the 3′ primers used for the second stepPCR carry a unique restriction endonuclease site at its flank. The 5′ VL(SEQ ID NO: 1) carried a Sac I site, while the 3′ VL (SEQ ID NO: 8) hadXba I site. The 5′ VH (SEQ ID NO: 9) and the 3′ VH primers carried Nco Iand Spe I sites, respectively. To eliminate an existing Sac I site fromthe constant region of the kappa chain, a point mutated Sac I site wasincorporated into the 3′ VL primer.

The first PCR was carried out in a final volume of 50 μl containing 10ng of template, 200 μM dNTPs and 5 units of Taq polymerase (Gibco BRL,Gaithersburg, Md.). The PCR mix contained 200 pmol of each of the 5′ and3′ primers; the latter being a mixture of degenerate primers. Thirtycycles of a denaturing step at 94° C. for 30 seconds, a primer annealingstep at 55° C. for 50 seconds, and a polymerization step at 72° C. for60 seconds were followed by a final primer extension step for 10 minutesat 72° C. The second PCR consisted of 30 cycles of denaturation (94° C.for 30 seconds), primer annealing (55° C. for 90 seconds) andpolymerization (72° C. for 90 seconds) followed by a final extension for10 minutes at 72° C.

Phagemid Vector

A phagemid vector pComb3H-SS (Barbas, C. F. and Burton, D. R. ColdSpring Harbor Laboratory Course on Monoclonal Antibodies FromCombinatorial Libraries, Cold Spring Harbor, N.Y., 1994) was used togenerate a combinatorial library of the mutated HuCC49V10 Fabs displayedon the surface of the filamentous phage M13. The SS designation is usedfor pComb3H vector when it carries a 1200 bp stuffer sequence in placeof Ig light chain (SS I), and a 300 bp sequence as a stuffer in place ofIg heavy chain (SS II). PComb3H-SS, a modified version of the originalpComb3 vector (Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978,1991), was obtained from Dr. Carlos Barbas of Scripps ResearchInstitute, LaJolla, Calif.

pComb3 contains both the origin of replication of the plasmid Co1E1 andorigin of replication of the filamentous bacteriophage f1. The plasmidcontains the gene for ampicillin (carbenicillin) resistance. Moreimportantly, it is designed to carry a number of unique and convenientrestriction endonuclease sites (FIG. 2). The restriction fragment EcoRI/Sac I located downstream from lac Z promoter carries a ribosomalbinding site and the outer membrane (omp A) leader sequence of E. coli.A sequence encoding an Ig light chain can be inserted as a Sac I/Xba Ifragment, immediately downstream from the omp A. Located 3′ to omp A isa Xba I/Nco I fragment that contains a stop codon for the translation ofthe light chain, a ribosomal binding site for the translation of thesecond protein from the bicistronic message, and a pel B leader peptideof E. carotovora. A sequence encoding the Fd fragment can be inserted asa Nco Spe I fragment, immediately downstream from the pel B leader. Thisis followed by a Spe I/Not I fragment that contains the carboxy-terminalpart of the gene III protein of phage M13 and two tandem stop codons.The carboxy-terminal part of gene III, which is fused to the Fd througha flexible linker, is located on a Spe I/Nhe I fragment. This fragmentcan be removed by cleavage with Spe I/Nhe I, followed by re-ligationwhich is possible because cleavage with the two enzymes generatesidentical cohesive ends. The leader peptides facilitate transport of theexpressed light chain and Fd fragments to the periplasm, where theleader peptides are proteolytically cleaved and the free light chainsand Fd fragments assemble into Fabs. The carboxyl-terminal half of geneIII serves a capping role in the morphogenesis of filamentous phages. Inthe presence of the helper phage, one Fab-gene III fusion proteinmolecule along with 3-4 molecules of the native gene III protein aredisplayed on the phage surface. The helper phage is essential forreplication and assembly of phage particles, because pComb3 is devoid ofall these genes. The helper phage, VCSM13, used here for the rescue ofthe phage particles carries a kanamycin resistance gene that facilitatesselection of bacteria infected with the helper virus. Arecombination-deficient strain of E. coli, XL1-Blue, carrying atransposon 10 in its F′ factor has been used to develop the phagedisplay libraries. The F′ factor is essential for the susceptibility ofbacteria to the male-specific phages. Transposon 10 carries atetracycline resistance gene.

Library Construction

To clone the light chain PCR products in pComb3H vector, the PComb3H-SSplasmid was simultaneously treated with 5 units of Sac I and 9 units ofXba I for each microgram of DNA in a reaction mixture of appropriatevolume containing the desired buffer. The reaction mixture was incubatedfor 3 hours at 37° C. The DNA was ethanol precipitated, pelleted, washedwith 70% ethanol, air dried and suspended in 100 μl of Tris-EDTA (TE)buffer (pH 8.0). To isolate the linearized vector, the DNA waselectrophoresed through 0.6% agarose gel. The gel containing a band ofapproximately 3.7 Kb DNA, visualized by ethidium bromide staining, wasexcised and the DNA was recovered from the agarose slice byelectroelution. The recovered DNA was ethanol precipitated, pelleted,washed, dried and resuspended in TE buffer as before. Similarly, the PCRproducts generated by using the light chain primers were treated withSac Xba I, and the purified Sac I/Xba I fragments were electrophoresedthrough a 2% agarose gel. The 750 bp DNA band was excised, and the DNAfragments were recovered from the gel by electroelution, as describedearlier. Multiple reactions were set up to ligate the Sac Xba Ilinearized vector and the Sac Xba I digested PCR products. Ligation wasperformed for 1 hour at room temperature, using a commercially availableligation kit (Gibco BRL, Gaithersburg, Md.). The ligation reactions werepooled and the DNA was ethanol precipitated, pelleted, washed, dried andresuspended in 15 μl of water. Electroporation-competent XL-1Blue cells(Stratagene, La Jolla, Calif.) were transformed with the ligated DNA byelectroporation. Using an electroporator (BioRad, Hercules, Calif.), apulse of 1700 volts at the field strength of 17 kV/cm was applied for 5milliseconds. A series of transformations were performed and pooledtogether. After adding SOC medium (Gibco BRL), the transformation mixwas incubated at 37° C. for 1 hour. Subsequently, Super Broth (GibcoBRL) containing 50 μg/ml of carbenicillin and 10 μg/ml of tetracyclinwas added and the culture was incubated at 37° C. on a shaker,overnight. The plasmid was isolated from the transformed cells, using amaxiprep kit (Qiagen, Valencia, Calif.). The isolated plasmid waslinearized by digestion with 3 units of Spe I and 9 units of Nco I perμg of the DNA. A procedure similar to that described for preparing SacI/Xba I linearized vector was used to prepare Nco Spe I linearizedplasmid. Similarly, the PCR products that were generated by using theheavy chain primers were digested with Nco I/Spe I and purified. Thepurified PCR products were inserted into the vector by ligation, and theligation mixture was used to transform XL-1Blue cells, byelectroporation. After adding tetracyclin- andcarbenicillin-supplemented Super Broth to the culture and incubating for1 hour at 37° C., 1 ml of VCSM13 helper phage (Stratagene) containingapproximately 10¹² pfus (plaque forming units) was added to the culture.After incubation for 2 hour, kanamycin (70 μg/ml) was added and theculture was shaken overnight at 37° C. Next morning, cells were spundown (4,000 rpm, 15 minutes, 4° C.) and the phage was precipitated fromthe supernatant by adding PEG-8000 and NaCl to the final concentrationof 4% and 3%, respectively, and incubating on ice for 30 minutes. Theprecipitate was collected by centrifugation (9,000 rpm, 20 minutes, 4°C.). The pellet was suspended in 2 ml of Tris borate saline (TBS)containing 1% BSA. The phage was titered by infecting XL-1Blue cells(0D₆₀₀=0.5) with serial dilutions of phage suspension, and plating theinfected cells on LB/carbenicillin plates.

Example 2 Selection of TAG-72-Binding HuCC49V10 Variants and Productionof Soluble Fabs

The phage library was screened and enriched for isolates binding toTAG-72. To that end, the library was subjected to multiple (7) rounds ofpanning. During each round, variants that specifically bind to TAG-72were selected and amplified. The selected variants were used as a sourceof phagemids that were isolated and genetically manipulated en mass toexpress soluble Fab molecules.

Panning

For panning, a modification of the procedure that has been describedearlier (Parmley and Smith Gene 73:305, 1988; Barbas et al., Proc. Natl.Acad. Sci. USA 88: 7978, 1991) was used. During each round of panning,variants that specifically bind to TAG-72 were selected and amplified.ELISA plates (Nalgene Nunc International, Rochester, N.Y.) were coatedovernight at 4° C. with 50 μl of TAG-72 positive bovine submaxillarymucin (BSM) (Type 1-S; Sigma, St Louis, Mo.) in D-PBS with calcium andmagnesium chloride (Gibco BRL), The amount of BSM was progressivelyreduced from 1.0 μg to 0.01 μg/well, with increasing rounds of panning.The wells were washed with water and blocked by incubating with milkblocking solution (KPL, Gaithersburg, Md.) at 37° C. for 1 hour. Fiftyμl of phage (0.15-5×10¹² pfus) suspended in 50 μl of milk diluentsolution (KPL) was pre-incubated at room temperature for 30 minutes,before it was added to the wells and incubated at 4° C. overnight. Afterremoving the milk/phage solution, the wells were washed by pipettingTBS/0.5% Tween 20 vigorously up and down. The washing cycles wereprogressively increased with increasing rounds of panning. Finally thephage was eluted by adding 50 μl of elution buffer (0.1 M HCl, pH2.2/BSA 1 mg/ml) and incubating for 10 minutes at room temperature. Theeluate was removed and neutralized with 43 μl of 1 M Tris base. Theeluted phage was used to infect growing XL-1 Blue cells, at roomtemperature for 15 minutes, and the virus was replicated in the presenceof helper phage VCSM13. Super Broth (SB) medium containing carbenicillinand tetracycline was added, and the culture was shaken at 37° C. for 1hour. Phage preparations and pannings were repeated, as describedearlier. After the final round of panning, the virus was harvested,precipitated and re-suspended in TBS/1% BSA.

The stringent condition of panning (decreasing amounts of BSM on theplate and increasing number of washing cycles with the higher rounds ofpanning) reduced the titer of the eluted phage from 1.2×10⁹ in the firstround to 1.3×10⁶ in the seventh round. The phage eluted from the seventhround was amplified to a titer of approximately 2×10¹³. Thus, asignificant enrichment of TAG-72 binding variants was achieved.

Preparation of Soluble Fab: Genetic Manipulation of Phagemid for SolubleFab Expression

The phage eluted from the last round of panning was used to infectlogarithmically growing XL-1Blue cells. The culture was grown in SBmedium containing carbenicillin (20 μg/ml) and tetracycline (10 μg/ml),overnight at 37° C. Cells were collected by pelleting and phagemid DNAwas isolated. The DNA was digested by treatment with Nhe I/Spe I toremove DNA fragment that encodes gene III. The enzyme treated DNA waselectrophoresed on a 0.6% agarose gel. The large DNA fragment was gelpurified, self-ligated and used to transform competent XL-1Blue cells.Transformation mixture was streaked on LB/carbenicillin plates. Afterincubating the plates o/n at 37° C., fifty individual colonies wereinoculated in 10 ml of SB medium containing 20 mM MgCl₂ and 50 μg/mlcarbenicillin. Cultures were grown at 37° C. for 6 hours, beforeisopropyl-β-D thiogalactopyranoside (IPTG) was added to a finalconcentration of 1 mM for the induction of the Fab expression. Theculture was then shifted to 30° C. and shaken overnight. Cells wererecovered by centrifugation. A part of the cell pellet was saved toisolate the phagemid for the subsequent sequence analysis of thevariants. The rest of the cell pellet was resuspended in 1 ml of PBS andlysed by four cycles of freezing in a dry ice-ethanol bath for 5 minutesand thawing in a 37° C. water bath. The cell debris was pelleted bycentrifugation at 15,000 rpm, and the supernatant was collected for theFab assay.

Example 3 Screening for High-Binding-Affinity Variants of HuCC49V10

Cell lysates from all 48 cultures of the XL-1Blue cells transformed bythe Fab constructs of pComb3H lacking the gene III fragment were testedfor the presence of Fab by ELISA and Western Blot analysis. The celllysates that were found positive were then screened for binding to theTAG-72 positive BSM by Surface Plasmon Resonance (SPR).

ELISA

Supernatants from each of the 48 cell lysates were screened to detectFab by ELISA assay that has earlier been described (Tamura et al., J.Immunol. 164:1432, 2000; Bei et al., J. Immunol. Methods 186: 245,1995). Individual wells of the 96-well polyvinyl microtiter plates werecoated with 0.1 μg (50 μl) of goat anti-human kappa (SouthernBiotechnology Associate, Inc., Bringham, Ala.). The plates were blockedwith 5% BSA in PBS for 1 hour at 37° C. and then washed with 1% BSA inPBS. Fifty μl of the supernatant to be tested was loaded in each well.After a 1 hour incubation at 37° C., plates were washed with 1% BSA inPBS. This was followed by adding 100 μl of 1:5,000 dilution (in 1% BSAin PBS) of peroxidase-conjugated goat anti-human IgG (Fab)₂ fragmentspecific antibody (Jackson Immuno Research Lab, West Grove, Pa.). Theplates were incubated for another hour at 37° C. They were washed priorto the addition of 100 μl of freshly prepared substrate (H₂O₂) mixedwith o-phenylene diamine hydrochloride as a chromogen (Sigma, St. Louis,Mo.). The colorimetric reaction was allowed to proceed for 10 min atroom temperature in the dark, before it was terminated by the additionof 50 μl of 4N H₂SO₄ per well. The absorbance was read at 490 nm.

Of the 48 lysates that were tested in duplicate, a great majority ofthem were positive, and many among them were strongly positive for theFab.

Western Blot Analysis

The presence of soluble Fab in cell lysates was also tested by WesternBlot. Forty μl of the supernatant from each of the 48 cell lysates waselectrophoresed on a 4-20% pre-cast SDS-polyacrylamide gel. Two sets ofSDS-PAGE were carried out, one under reducing and the other undernon-reducing conditions. The proteins were electro-blotted on to theImmobilon-P membrane (Millipore, Bedford, Mass.) according to theinstructions of the manufacturer. The blotted membrane was saturatedwith 5% milk for 1 hour, washed, dried and then probed withperoxidase-conjugated goat anti-human IgG (Fab)₂ fragment specificantibody (Jackson ImmunoResearch Lab, West Grove, Pa.). The membrane waswashed several times with TBS/0.05% Tween 20, before ECL Westernblotting detection kit (Amersham Pharmacia Biotech UK Limited,Buckinghamshire, UK) was used to detect the protein band.

Western blots that were done after electrophoresis under non-reducingconditions showed a band of approximately 55 kD, a size in conformitywith that of a Fab molecule. Similarly, Western blotting done followingSDS-PAGE under reducing conditions yielded a band of 27-28 kD, a sizeexpected of the Fd fragment. The Fab could be detected in 47 of the 48cell lysates that were tested, albeit the degree of the intensity of theband varied.

Screening of Fabs for Their TAG-72 Binding Affinity by SPR

The expressed Fab molecules were screened for their immunoreactivity tothe TAG-72 positive BSM, using BIAcore X instrument (Biacore,Piscataway, N.J.) for SPR measurements. All samples were run induplicate over a sensor chip immobilized with 500 Resonance Units (RU)of BSM. Another sensor chip immobilized with 500 RU of BSA was used as areference. Proteins were immobilized on carboxymethylated dextran CM5chips (Biacore) by amine coupling using standard procedures (Johnsson etal., Anal. Biochem., 198: 268, 1991; Schuck et al., 1999 Measuringprotein interactions by optical biosensors. In: J. E. Coligan, B. M.Dunn, H. L. Ploegh, D. W. Speicher, and P. T. Wingfield (Eds.) CurrentProtocols in Protein Science. John Wiley & Sons, New York, Vol. 2, p.20.2.1.) One hundred μl of each sample was applied at a flow rate of 20μl/min and the dissociation was observed for 300 seconds. After thesamples were washed with the running buffer, the surfaces wereregenerated with 1 M CAPS buffer. The BIAeval 3.0.2 program was used toanalyze the data. The BSA sensorgram was subtracted from thecorresponding BSM sensorgram and the Langmuir dissociation model wasused to evaluate the off rate (k off).

Supernatants from all 48 cell lysates were analyzed for their reactivityto BSM. Since the Fabs were not purified, they were evaluated for theirdissociation rates only. Fabs derived from the murine CC49, HuCC49V10and human IgG (HuIgG) were included as controls. The dissociation ratesof only 6 isolates were lower than that of HuCC49V10 (Table 5). Theywere characterized further, because these isolates are likely to havehigher affinity for TAG-72.

TABLE 5 Dissociation rates of Fabs by Surface Plasmon Resonance IsolateK_(off) (1/s) HuCC49V10-12 2.20 × 10⁻⁴ HuCC49V10-7 2.58 × 10⁻⁴HuCC49V10-14 9.14 × 10⁻⁴ HuCC49V10-15 1.21 × 10⁻⁴ HuCC49V10-10 2.50 ×10⁻³ HuCC49V10-13 4.34 × 10⁻⁴ mCC49 1.04 × 10⁻⁴ HuCC49V10 1.07 × 10⁻³HuIgG 1.38 × 10⁻²

The phagemids prepared from the cell pellets of the six isolates wereused for the sequencing of the inserts encoding the variable regions ofthe light and heavy chain. DNA sequencing was carried out by the methodof dideoxy-mediated chain termination. Amino acid sequences deduced fromthe nucleotide sequences showed substitutions in LCDR3 of all the sixvariants. These substitutions were limited to the positions 91, 93 and94. Whereas, only one variant showed substitutions in HCDR2 (positions52 and 58). Two variants showed inadvertent mutation in position 27b ofthe LCDR1 (Table 2).

Example 4 Expression of HuCC49V10 Variants in Insect Cells; Purificationand Characterization of the Expressed Antibodies

For further characterization of the variants, they were expressed ininsect cells as whole antibodies, rather than as Fab fragments. To thatend, expression constructs of the genes encoding the heavy and lightchains of the variants were made in vectors containing promoters thatare functional in insect cells. The variant antibodies were purified andstudied for their relative antigen-binding affinity and their ability tobind to a cell surface antigen.

Expression Constructs of the Heavy and Light Chain Genes of the VariantAntibodies

Two different vectors, pIZ/V5-His (FIG. 3A) and pIB/V5-His (FIG. 3B)(Invitrogen, Carlsbad, Calif.), carry the baculovirus immediate earlypromoter OpIE2 that drives the expression of heterologous proteins inlepidopteran insect cells, without requiring viral factors for itsactivation. A multiple cloning site located downstream from thepromoter, in both vectors, facilitates cloning of the gene of interestto be expressed constitutively. A polyadenylation sequence placedimmediately 3′ to the multiple cloning site ensures efficienttranscription termination and polyadenylation of mRNA. One vector,pIZ/V5-His, carries the Zeocin resistance gene, while the other,pIB/V5-His, carries the blasticidin resistance gene. These genes areused for selecting stable transfectants of the insect cell line.

The sequences encoding the light chain of the variants were assembled inthe pIZ/V5-His vector. A series of genetic manipulations were requiredto provide a leader sequence for each of the genes encoding the lightchain. Essentially, a 538 bp BsmA I/Blp I DNA fragment carrying theLCDR3 and/or LCDR1 mutation(s) and extending into the constant regionwas PCR amplified from each of the variants. This DNA fragment replacedthe corresponding sequence from a pBluescript construct of the variantHuCC49V10-5, which carried the light chain gene of HuCC49V10 along withits leader sequence. The construct was digested with Sma I, for which aunique site was located immediately 3′ to the insert, an Xba I linkerwas ligated to the DNA ends, and the insert was lifted as a Hind III/XbaI fragment of approximately 1 Kb. The insert was cloned downstream fromthe OpIE2 promoter at the Hind III/Xba I site.

The heavy chain expression constructs were made in the pIB/V5-Hisvector. Since only one (variant HuCC49V10-13) of the six variants showedany mutations in the HCDR2, the heavy chain construct of the variantHuCC49V10-8, which carried the heavy chain gene of the HuCC49V10 alongwith its leader sequence, was paired with the light chain constructs ofthe variants (variants HuCC49V10-7, HuCC49V10-10, HuCC49V10-12,HuCC49V10-14 and HuCC49V10-15) for the production of the variantantibodies. The gene encoding the heavy chain along with its leader wasexcised from the pBSc construct of the variant HuCC49V10-8 as a HindIII/Xho I fragment. The insert was cloned at the Hind III/Xho I site,downstream of the OpIE2 promoter in pIB/V5-His vector.

Construction of the expression vector containing the heavy chain ofvariant HuCC49V10-13 in the pIB/V5-His vector, while incorporating theeukaryotic leader sequence between the promoter and the gene, requiredsome genetic manipulation. Essentially, an 85 bp Bcl I/Sca I fragment ofvariant HuCC49V10-8 was replaced with the corresponding DNA fragment ofvariant HuCC49V10-13 that carried the mutations in HCDR2. Themanipulated insert was lifted from the construct as a Hind III/Xho Ifragment and cloned in pIB/V5-His vector, downstream from the promoter.

Production of the Variant Antibodies and Their Immunoreactivity

To develop transfectomas secreting the HuCC49 variants,serum-free-adapted Sf9 insect cells (Gibco BRL) were used. Two millioninsect cells plated in each well of a 6-well plate were co-transfectedwith 10 μg each of the pIZ/V5-His-light chain and pIB/V5-His-heavy chainconstructs, using Insectin-Plus Liposomes (Invitrogen) to mediate thetransfections. After four days, the culture supernatants were harvestedand tested, by ELISA, for Ig secretion and the reactivity of thesecreted antibody to TAG-72. For isolating stable transfectants,selection medium containing 200 μg/ml of zeocin and 50 μg/ml ofblasticidin was used. For ELISA assays, four-day harvests of culturesupernatants were collected and frozen at −20° C., prior to use.

The ELISA assay for monitoring Ig production was carried out by aprocedure described earlier (Example 3). To test the reactivity of thesecreted antibody to TAG-72, 1 μg/well of BSM was coated on theindividual wells of the 96-well polyvinyl microtiter plates. Aftersaturation of the plates for 1 hour at 37° C. with milk blockingsolution (KPL), 50 μl of diluted culture supernatants were added to thewells, in duplicate, followed by incubation at 37° C. for 1 hour. Aftera cycle of washings with the washing solution (KPL), 100 μl ofperoxidase-conjugated anti-human IgG (Fcγ-fragment specific), diluted1:3000 in milk diluent solution (KPL), was added to the plates and theincubation continued for an additional hour at 37° C. They were washedprior to the addition of 100 μl of TMB peroxidase substrate (KPL). Thecolorimetric reaction proceeded for 10 minutes at room temperature,before the addition of the stop solution (KPL). The absorbance was readat 450 nm.

Results of the ELISA of the culture supernatants for IgG showed that allthe variants produced antibody. When the culture supernatants wereassayed for their reactivity to TAG-72, it became evident that theantibodies produced by the variants were specific to TAG-72. When theELISA was carried out using serially diluted supernatants, the resultsof the assay suggested that the antigen-binding reactivity of, at leasttwo variant antibodies, HuCC49V10-14 and HuCC49V10-15, was eithercomparable to or exceeded that of the parental HuCC49V10, while thereactivity of variant HuCC49V10-13 was convincingly lower than that ofHuCC49V10. Variant HuCC49V10-13 was not included in further studies.

Purification of the Variant Antibodies and Their SDS-PAGE Analysis

The supernatants collected from the cultures of the transfectomasproducing the variant antibodies were centrifuged at 2,000×g for 10minutes to remove cellular debris and loaded on a protein G agarosecolumn (Gibco BRL). 0.1 M glycine hydrochloride pH 2.5 was used to elutethe proteins bound to the column. The pH of the eluted material wasimmediately adjusted to 7.4 with 1.0 M Tris pH 8.0. The proteins wereconcentrated using a Centricon 30 (Amicon, Beverly, Mass.) and dialyzedin PBS buffer using Slide-A-Lyzer cassette (Pierce, Rockford, Ill.). Theprotein concentration was determined by the method of Lowry (Lowry etal., J. Biol. Chem. 193: 265,1951). The concentration of the variantantibodies ranged between 2-5 μg/ml of the culture supernatant. Thepurity of the eluted proteins was evaluated by SDS-PAGE, under reducingand non-reducing condition, using pre-cast 4-20% Tris-glycine gel(Novex, San Diego, Calif.) and Coomassie blue staining (Novex)visualization. Under non-reducing conditions (FIG. 4A) a protein band ofapproximately 160 kD was seen, while the reducing condition (FIG. 4B)yielded two protein bands of approximately 50-55 kD and approximately25-28 kD, the sizes expected of the heavy and light chains of an IgGmolecule.

Competition Radioimmunoassay (RIA)

The relative antigen-binding affinity of the variant antibodies wasdetermined using competition RIA, as described earlier (Iwahashi et al.,Mol. Immunol. 36:1079, 1999; Tamura et al., J. Immunol. 164: 1432,2000). Murine CC49, HuCC49 and HuIgG were included in the assay aspositive and negative controls. Twenty five μl of serial dilutions ofthe antibodies, re-suspended in 1% BSA in PBS, were added to microtiterplates containing 10 ng of BSM saturated with 5% BSA in PBS.¹²⁵I-labeled HuCC49 (100,000 cpm in 25 μl of 1% BSA in PBS) was thenadded to each well. The assay was set up in triplicate. After anovernight incubation at 4° C., the plates were washed and counted in aγ-scintillation counter.

The results of the competition assay (FIG. 5) show that all theantibodies, except the HuIgG control, were able to completely inhibitthe binding of ¹²⁵I-labeled HuCC49 to BSM. The competition profiles ofthe variants HuCC49V10-7 and HuCC49V10-12 were shifted to the right,while the profile of HuCC49V10-14 was shifted only slightly and that ofHuCC49V10-15 considerably to the left of the competition profile of theparental antibody HuCC49V10. Competition profiles, shown in FIG. 5, wereused to calculate the amount of each unlabeled competitor required for50% inhibition of the binding of ¹²⁵I-labeled HuCC49 to BSM (Table 6).Compared to 150 ng of HuCC49V10, 220 ng and 350 ng of variantsHuCC49V10-7 and HuCC49V10-12, respectively, were needed. In contrast, 92ng of HuCC49V10-14 and only 58 ng of HuCC49V10-15 were required for 50%inhibition of the binding of ¹²⁵I-labeled HuCC49 to BSM. Thus,HuCC49V10-15 is approximately 3-fold better than that of HuCC49V10antigen binding. The relative affinity constants (K_(a)) werecalculated, from a range of competition experiments, using amodification of the Scatchard methods (Frankel and Gerhard, Gene 16:101,1979). The K_(a) values for HuCC49V10-14 were only comparable, whilethose for HuCC49V10-15 were approximately 50% higher than that ofHuCC49V10.

TABLE 6 Relative affinity binding of CC49 antibodies Amount needed for50% inhibition of CC49 antibody the binding of ¹²⁵I-labeled HuCC49 toBSM (ng) mCC49: 25 HuCC49: 78 HuCC49V10: 150 HuCC49V10-7: 220HuCC49V10-12: 350 HuCC49V10-14: 92 HuCC49V10-15: 58

Flow Cytometric Analysis

Flow cytometric analysis was used to measure the binding of theHuCC49V10 variants to the TAG-72 expressed on the cell surface of a Tcell line, Jurkat. The procedure for FACS analysis has been described(Guadagni et al., Cancer Res. 50:6248, 1990). In addition to the isotypematched antibody, HuIgG, used as a negative control, HuCC49 and chimericCC49 were included as positive controls. To evaluate the ability of thevariants to bind to cell-surface TAG-72, 1×10⁶Jurkat cells wereresuspended in cold Ca⁺⁺ and Mg⁺⁺ free Dulbecco's PBS and incubated withthe antibody to be tested for 30 minutes on ice. After one washingcycle, the cell suspension was stained with FITC-conjugated mouseanti-human antibody (Pharmingen) for 30 minutes on ice. A second washingcycle was performed before the samples were analyzed with a FACScan(Becton Dickinson, Mountain View, Calif.) using CellQuest for Macintosh.Data from the analysis of 10,000 cells were obtained.

Different concentrations of antibodies were used to compare theirbinding to the Jurkat cells expressing cell surface TAG-72. When 1.0 μgof each antibody was used, the percentage of gated cells, calculatedafter exclusion of irrelevant binding, was 27.8 for HuCC49V10, while forthe variants HuCC49V10-14 and HuCC49V10-15, it was 43.7 and 68.1,respectively. Thus, the two variants show significantly better bindingto the cells displaying TAG-72 on their surface. In contrast, thebinding of the variants HuCC49V10-7 and HuCC49V10-12 was comparable tothat of the parental HuCC49V10 (FIG. 6).

Example 5 Sera Reactivity of HuCC49V10 Variants

To assess the potential immunogenicity of the HuCC49V10 variants inpatients, the variants were tested for their reactivity to sera storedfrom the adenocarcinoma patients in a phase I clinical trial (Mulliganet al., Clin. Cancer Res. 1: 1447, 1995). Patients in this clinicaltrial were administered ¹⁷⁷Lu-labeled murine CC49 and were found to haveanti variable region, including anti-idiotypic, antibodies to CC49(Iwahashi et al., Mol. Immunol. 36: 1079, 1999; Tamura et al., J.Immunol. 164: 1432, 2000). Sera reactivity was determined by a highlysensitive Surface Plasmon Resonance (SPR)-based competition assay. Thisassay involves the use of a device (BIAcore X instrument) that monitorsbinding of the sera anti-idiotypic (or anti variable region) antibodiesto HuCC49, and the inhibition of this binding by the variants. IC₅₀, theconcentration of the competitor antibody required for 50% inhibition ofthe binding of the HuCC49 to the patient's serum was calculated byplotting the percent inhibition as a function of the competitorconcentration. A higher IC₅₀ indicates a decreased reactivity to theserum suggesting potentially reduced immunogenicity of the competitorantibody (HuCC49 variant) in patients.

Immunoadsorption of Patients Sera

Sera from patient EA and DS from the phase I clinical trial were used tocompare the reactivity of the variants HuCC49V10-14 and HuCC49V10-15 tothat of the parental HuCC49V10. The sera, however, contain circulatingTAG-72 antigen and anti-murine Fc antibodies that might interfere withthe binding of the HuCC49 and its variants to the sera anti-idiotypic(anti variable) antibodies. To overcome this difficulty, TAG-72 andantibodies to murine Fc were removed from the sera by immunoadsorptionprior to checking the sera reactivity. The procedure forimmunoadsorption has been described (Iwahashi et al., Mol. Immunol. 36:1079, 1999; Tamura et al., J. Immunol. 164: 1432, 2000). Essentially, amurine antibody, CC92, which reacts with an epitope of TAG-72 distinctfrom the one recognized by CC49 (Kuroki et al., Cancer Res. 50: 4872,1990) was coupled to Reactigel (HW65F; Pierce, Rockford. IL) (Hearn etal., J. Chomatogr. 185: 463, 1979). Serum was added to an equivalentvolume of the CC92 gel (wet-packed volume) and incubated overnight at 4°C. with end-over-end rotation. The samples were centrifuged at 1,000×gfor 5 minutes and the supernatant was saved and stored.

SPR-Based Competition Assay

To test the sera reactivities of antibodies, SPR measurements were donewith the BIAcore X instrument (described in Example 3) usingcarboxymethylated dextran chips CM5 (BIAcore, Piscataway, N.J.).Proteins were immobilized on the CM5 chips by amine coupling (Johnssonet al., Anal. Biochem. 198: 268, 1991). The dextran layer of the sensorchip was activated by injecting 35 μl of a mixture of N-ethyl-N % (3dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimideat a flow rate of 5 μl/min. Proteins diluted in 10 mM sodium acetatebuffer (pH 5.0) at a concentration of 100 μg/ml were then injected untilsurfaces of 5000 resonance units (RUs) were obtained. The remainingreactive groups on the surfaces were blocked by injecting 35 μl of 1 Methanolamine (pH 8.5).

To compare the sera reactivity of HuCC49, HuCC49V10 and its variantsHuCC49V10-14 and HuCC49V10-15, competition experiments were done at 25°C. on a sensor chip containing HuCC49 in flow cell 1 and rabbit gammaglobulin (BioRad, Hercules, Calif.) in flow cell 2 as a reference. Arecently developed sample application technique was used (Abrantes etal., Anal. Chem. 73: 2828, 2001), in which the microfluidics control ofthe instrument was replaced by an externally installedcomputer-controlled syringe pump with stepping motor (model 402 fromGilson Inc., Middleton, Wis.). A tubing was inserted into the open portof the connector block in order to serve as an inlet port through whichthe sample can be aspirated, and the port previously designated asrunning buffer inlet was connected to the syringe pump (Abrantes et al.,Anal. Chem. 73: 2828, 2001). The computer-controlled aspiration made itpossible to use small sample volumes. Typically, the microfluidicssystem was rinsed and filled with running buffer (10 mM HEPES (pH 7.4),150 mM NaCl, 3 mM EDTA and 0.005% Tween 20). This was followed bysequential aspiration of 2 μl of air, 0.3 μl of sample (preabsorbedserum±antibodies as competitors), 2 μl of air, 5-6 μl of sample, 2 μl ofair, 0.3 μl of sample, and 2 μl of air into the inlet tubing at a rateof 20 μl/min. The sample was centered across the sensor surface and anoscillatory flow was applied at a rate of 20 μl/min. This flow ensuresefficient mass transfer of the sample to the surface and allows for avery long contact time without net displacement of the sample. Thebinding was measured for 1000 seconds. After the unbound samples wereremoved from the surfaces by washing with running buffer using a flowrate of 100 μl/min, the surfaces were regenerated with a one-minuteinjection of 10 mM glycine (pH 2.0). The percent binding at eachantibody concentration was calculated as follows: % binding=[slope ofthe signal obtained with competitor (serum+antibody)/slope of the signalobtained without competitor (serum only)]×100.

FIG. 7 shows the competition profiles generated by HuCC49 and differentvariants when they were used to compete with the HuCC49 immobilized onthe sensor chip for binding to the anti-idiotypic (anti variable)antibodies to CC49 present in the sera of the patients EA and DS. Forserum from patient DS, approximately one micromole of the variantHuCC49V10 is required for 50% inhibition of the binding of HuCC49 to theserum, while the variants HuCC49V10-14 and HuCC49V10-15 do not show anysignificant inhibition at this concentration. For serum from patient EA,approximately 20 nanomoles of the HuCC49V10 is needed for 50% inhibitionof the binding of HuCC49 to the serum, whereas one micromole of each ofthe HuCC49V10-14 and HuCC49V10-15 variants cause less than 40%inhibition. Thus, the two mutants showed not only significantly higherantigen binding affinity than that of HuCC49V10, but they also showedmuch lower reactivity to sera from patients who showed an anti-idiotypicresponse to the parental CC49 antibody.

The improved affinity and the minimal sera reactivity of the variantsHuCC49V10-14 and HuCC49V10-15 make them potentially much better clinicalreagents than the variant HuCC49V10.

Example 6 HuCC49V10-14 and HuCC46V10-15 Testing in Patients Patients andSample Collection

Patients with recurrent metastatic adenocarcinoma are assessed todetermine the maximum tolerated dose of intravenously administered¹⁷⁷Lutetium radiolabeled HuCC49V10-14 and ¹⁷⁷Lutetium radiolabeledHuCC49V10-15 (Mulligan, (1995) Clin. Cancer Res. 1:1447-1454).Adenocarcinoma patients are given a test dose of 0.1 mg (intravenousbolus) of HuCC49V10-14 or HuCC49V10-15 and are observed for 30 minutesprior to administration of the ¹⁷⁷Lu-labeled HuCC49V10-14 or¹⁷⁷Lu-labeled HuCC49V10-15. The radiolabeled antibodies are given as anintravenous infusion over the course of a one hour time interval. Bloodsamples are collected prior to and at the end of the infusion, as wellas 0.5, 1 and 2 hours following the completion of the infusion. Inaddition, blood samples are collected daily over the subsequent 7 days.Patients return for a follow-up examination at 3, 6 or 8 weeks. Bloodsamples are again collected during these visits. Sera are separated andstored at −20° C.

Determination of Patient Humoral Response

The sera from the patients are evaluated for the presence of humananti-murine antibodies (HAMA) in response to radiolabeled HuCC49V10-14or HuCC49V10-15 using the SPR-based assay described in Example 5, above.The sera is pre-absorbed with a CC92 monoclonal antibody that recognizesan epitope of TAG-72 which is different from the epitope recognized bythe humanized CC49 monoclonal antibody. Pre-absorption using the CC92antibody removes circulating TAG-72 from the sera. To monitor thesera-reactivity of the anti-variable antibodies in the pre-absorbedsera, HuCC49V10-14 or HuCC49V10-15 is coated on the surface of flow cell1 and a reference protein (HuIgG1, bovine serum albumin, or rabbit gammaglobulin) is immobilized on the surface of flow cell 2. A small, knownvolume of a patient serum sample us applied to each flow cell using therecently developed sample application technique described in Example 5(Abrantes et al., Anal. Chem. 73:2828, 2001). Sensograms to flow cell 1and flow cell 2 are generated and the response difference between thetwo cells is plotted for each serum sample, thus providing a measure ofthe anti-variable region response against HuCC49V10-14 or HuCC49V10-15in each particular serum sample. Results indicate that the patients'sera have a minimal anti-variable region response against theHuCC49V10-14 and HuCC49V10-15 antibodies.

This disclosure provides humanized CC49 monoclonal antibodies. Thedisclosure further provides methods of diagnosing and treating tumorsusing these humanized CC49 antibodies. It will be apparent that theprecise details of the methods described may be varied or modifiedwithout departing from the spirit of the described invention. We claimall such modifications and variations that fall within the scope andspirit of the claims below.

1. A method for determining the immunogenicity of a variant humanizedCC49 antibody, comprising: contacting anti-idiotypic antibodies with aparental humanized CC49 antibody and with the variant humanized CC49antibody; and determining the ability of the anti-idiotypic antibodiesto bind the variant humanized CC49 antibody and the parental humanizedCC49 antibody; wherein an at least five-fold higher molar concentrationof the variant humanized CC49 antibody than the molar concentration ofthe parental humanized antibody being required to elicit 50% inhibitionof the parental humanized CC49 antibody binding to the anti-idiotypicantibodies indicates that the variant humanized CC49 is minimallyimmunogenic, and wherein the parental humanized CC49 antibody comprisesone of: (a) four variable light framework regions and four variableheavy framework regions of a human antibody; a light chaincomplementarity determining region (L-CDR)1, a L-CDR2, a L-CDR3, a heavychain complementarity determining region (H-CDR)1, a H-CDR2, and aH-CDR3; a non-conservative substitution of a first residue at position91, wherein the first residue is in the L-CDR3 of the antibody; and asubstitution of a second residue at position 27b, wherein the secondresidue is in the L-CDR of the antibody; wherein the non-conservativesubstitution of the first residue at position 91 is a tyrosine toproline substitution, the substitution of the second residue at position27b is a valine to leucine substitution, the L-CDR1, L-CDR2, L-CDR3,H-CDR1, H-CDR2, and H-CDR3 are a HuCC49V10 antibody L-CDR1, L-CDR2,L-CDR3, H-CDR1, H-CDR2, and H-CDR3, respectively, and the parentalhumanized CC49 antibody has a high binding affinity for TAG-72 and isminimally immunogenic, as compared to the HuCC49V10 antibody, depositedas ATCC Accession No. PTA-5416; (b) a light chain complementaritydetermining region (L-CDR)1, a L-CDR2, and a L-CDR3, a heavy chaincomplementarity determining region (H-CDR)1, a H-CDR2, and a H-CDR3, allfrom the HuCC49V10 antibody; wherein the L-CDR3 of the parentalhumanized CC49 antibody comprises a non-conservative amino acidsubstitution at position 91 and has a high binding affinity for TAG-72,as compared to the HuCC49V10 antibody; (c) humanized CC49 antibodydeposited as ATCC Accession number PTA-4182; or (d) humanized CC49antibody deposited as ATCC Accession number PTA-4183.
 2. The method ofclaim 1, wherein the parental humanized CC49 antibody comprises fourvariable light framework regions and four variable heavy frameworkregions of a human antibody; a light chain complementarity determiningregion (L-CDR)1, a L-CDR2, a L-CDR3, a heavy chain complementaritydetermining region (H-CDR)1, a H-CDR2, and a H-CDR3; a non-conservativesubstitution of a first residue at position 91, wherein the firstresidue is in the L-CDR3 of the antibody; and a substitution of a secondresidue at position 27b, wherein the second residue is in the L-CDR ofthe antibody; wherein the non-conservative substitution of the firstresidue at position 91 is a tyrosine to proline substitution, thesubstitution of the second residue at position 27b is a valine toleucine substitution, the L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, andH-CDR3 are a HuCC49V10 antibody L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2,and H-CDR3, respectively, and the parental humanized CC49 antibody has ahigh binding affinity for TAG-72 and is minimally immunogenic, ascompared to the HuCC49V10 antibody, deposited as ATCC Accession No.PTA-5416.
 3. The method of claim 1, wherein the parental humanized CC49antibody comprises: a light chain complementarity determining region(L-CDR)1, a L-CDR2, and a L-CDR3, a heavy chain complementaritydetermining region (H-CDR)1, a H-CDR2, and a H-CDR3, all of theHuCC49V10 antibody, wherein the L-CDR3 of the parental humanized CC49antibody or of an antigen binding fragment of the parental humanizedCC49 antibody comprises a non-conservative amino acid substitution atposition 91 and has a high binding affinity for TAG-72, compared to theHuCC49V10 antibody.
 4. The method of claim 3, wherein thenon-conservative substitution is a tyrosine to proline substitution. 5.The method of claim 1, wherein the parental humanized CC49 antibodycomprises: four variable light framework regions and four variable heavyframework regions of a human antibody; a light chain complementaritydetermining region (L-CDR)1, a L-CDR2, and a L-CDR3 of the HuCC49V10antibody, a heavy chain complementarity determining region (H-CDR)1, aH-CDR2, and a H-CDR3 all from the HuCC49V10 antibody; a non-conservativesubstitution of a residue at position 91 in the L-CDR3 of the antibody;and a substitution of a residue at position 27b of L-CDR1 of theantibody; wherein the parental humanized CC49 antibody has a highbinding affinity for TAG-72 and is minimally immunogenic, compared tothe HuCC49V10 antibody.
 6. The method of claim 5, wherein thesubstitution at position 91 is a proline to tyrosine substitution andthe substitution at position 27b in the parental humanized CC49 antibodyis a valine to leucine substitution.
 7. The method of claim 1, whereinthe parental humanized CC49 antibody is the antibody deposited as ATCCAccession number PTA-4182.
 8. The method of claim 1, wherein theparental humanized CC49 antibody is the antibody deposited as ATCCAccession number PTA-4183.
 9. The method of claim 1, wherein an at leastten-fold higher molar concentration of the variant humanized CC49antibody than the molar concentration of the parental humanized antibodybeing required to elicit 50% inhibition of the parental HuCC49 bindingto the anti-idiotypic antibodies indicates that the variant humanizedCC49 is minimally immunogenic.
 10. The method of claim 1, wherein an atleast 25-fold higher molar concentration of the variant humanized CC49antibody than the molar concentration of the parental humanized antibodybeing required to elicit 50% inhibition of the parental HuCC49 bindingto the anti-idiotypic antibodies indicates that the variant humanizedCC49 is minimally immunogenic.
 11. The method of claim 1, furthercomprising: contacting the anti-idiotypic antibodies with murine Fcprior to contacting the anti-idiotypic antibodies with the parentalHuCC49 antibody and the variant HuCC49 antibody.
 12. The method of claim1, further comprising: determining reactivity of the variant humanizedCC49 antibody to serum from a subject, wherein the subject has beenadministered an effective amount of the variant humanized CC49 antibody.13. The method of claim 12, wherein determining the serum reactivitycomprises surface plasmon resonance.
 14. The method of claim 1, furthercomprising: determining the binding affinity of the variant humanizedCC49 antibody to TAG-72, bovine submaxillary mucin, or both.
 15. Themethod of claim 14, wherein determining the binding of the varianthumanized CC49 antibody comprises the use of a competitionradioimmunoassay.
 16. The method of claim 14, wherein determining thebinding of the variant humanized CC49 antibody comprises the use offluorescent activated cell sorting.
 17. The method of claim 1, whereinthe variant humanized CC49 antibody is labeled.
 18. The method of claim1, wherein the label is a radiolabel, an enzyme, an enzyme substrate, ora fluorescent agent.
 19. The method of claim 1, wherein the varianthumanized CC49 antibody binds TAG-72 with a K_(D) of at least 1.2×10⁻⁸.20. An isolated host cell transformed with a vector, wherein the vectorencodes: (a) four variable light framework regions and four variableheavy framework regions of a human antibody; a light chaincomplementarity determining region (L-CDR)1, a L-CDR2, a L-CDR3, a heavychain complementarity determining region (H-CDR)1, a H-CDR2, and aH-CDR3; a non-conservative substitution of a first residue at position91, wherein the first residue is in the L-CDR3 of the antibody; and asubstitution of a second residue at position 27b, wherein the secondresidue is in the L-CDR of the antibody; wherein the non-conservativesubstitution of the first residue at position 91 is a tyrosine toproline substitution, the substitution of the second residue at position27b is a valine to leucine substitution, the L-CDR1, L-CDR2, L-CDR3,H-CDR1, H-CDR2, and H-CDR3 are a HuCC49V10 antibody L-CDR1, L-CDR2,L-CDR3, H-CDR1, H-CDR2, and H-CDR3, respectively, and the parentalhumanized CC49 antibody has a high binding affinity for TAG-72 and isminimally immunogenic, as compared to the HuCC49V10 antibody, depositedas ATCC Accession No. PTA-5416; (b) a light chain complementaritydetermining region (L-CDR)1, a L-CDR2, and a L-CDR3, a heavy chaincomplementarity determining region (H-CDR)1, a H-CDR2, and a H-CDR3, allfrom the HuCC49V10 antibody; wherein the L-CDR3 of the parentalhumanized CC49 antibody comprises a non-conservative amino acidsubstitution at position 91 and has a high binding affinity for TAG-72,as compared to the HuCC49V10 antibody; (c) humanized CC49 antibodydeposited as ATCC Accession number PTA-4182; or (d) humanized CC49antibody deposited as ATCC Accession number PTA-4183.